-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
MECHANISMS OF LIVER PROGENOTOR CELL NICHE
ACTIVATION IN CANINE LIVER DISEASE
By; Drs. D.H. Loopstra
Studentnr.; 0248568
Timeframe; Oct-Dec ‘08
Supervisor; B.A. Schotanus
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Contents

Abstract
3

Introduction
o Etiology of canine hepatitis
o Histological changes in canine hepatitis
o Liver tissue repair
o Signalling pathways
o Notch signalling pathway
o Wnt signalling pathway
o Summary of previous research
4

Aim of the research project
9

Materials and Methods
o Immunohistochemistry
o Paraffin embedded tissue versus frozen tissue material
o Immunohistochemistry on paraffin-embedded liver tissue
o Immunohistochemistry on frozen liver tissue
o Liver tissue samples used
10
Results
o Paraffin-embedded liver tissue
o Frozen liver tissue
13

Discussion
21

Acknowledgements
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
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 References
24

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Attachments
o A; Paraffin-embedded liver tissue, Notch 1 protocol- and results table
o B; Paraffin-embedded liver tissue: β catenin protocol- and results table
o C; Stainingprotocol for Notch 1 (paraffin-embedded tissue)
o D; Stainingprotocol for β catenin (paraffin-embedded tissue)
o E: Stainingprotocol for CK7 (frozen tissue)
o F; Stainingprotocol for Notch 1 (frozen tissue)
o G; Stainingprotocol for β catenin (frozen tissue)
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Abstract
In this research project the liver specific progenitor cell (LPC) and its signalling
pathways were studied in the dog, by the use of immunohistochemistry. LPC’s play
an important role in the ability of the liver to regenerate in response to tissue
damage. After activation, LPC’s proliferate (then called reactive ductules) and can
differentiate into hepatocytes or cholangiocytes in order to restore the liver’s
physiological functions. Much is still unknown about this activation and the
signalling pathways involved in this process, making it an interesting subject for
research.
In this research project we were particularly interested in the Notch and Wnt
signalling pathways, since their involvement in the activation of human and rat
LPC’s has recently been suggested [14][8].
Initially, immunohistochemistry was performed on paraffin-embedded liver tissue
samples. Results showed that the use of paraffin-embedded liver tissue wasn’t
satisfying, since outcome of many different staining protocols used varied from
negative staining to such high background staining that no conclusions could be
drawn from these stainings.
In addition, immunohistochemistry was performed on frozen liver tissue samples of
different types of liver injury. Results showed increased staining of ß-catenin and
thus involvement of Wnt in the proliferation of LPC’s and their differentiation into
hepatocytes in acute hepatitis, active cirrhosis, PPVH and possibly extrahepatic
bile duct obstruction. Increased staining and thus involvement of Notch-1 in the
differentiation of LPC’s into cholangiocytes was seen mainly in active cirrhosis and
PPVH. This supports current ideas that Wnt is mainly involved in the proliferation
of LPC’s and differentiation into hepatocytes and that Notch-1 is mainly involved in
the differentiation of LPC’s into cholangiocytes.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Introduction
In veterinary medicine, canine liver disease is a common phenomenon. Fortunately,
the liver possesses an extra-ordinary way to respond to tissue damage;
regeneration. Liver regeneration, having presumably evolved to protect animals in
the wild from the catastrophic results of liver loss caused by food toxins, has been
an object of curiosity for many years. The ancient Greeks recognized liver
regeneration in the myth of Prometheus. Having stolen the secret of fire from the
gods of Olympus, Prometheus was condemned to having a portion of his liver eaten
daily by an eagle. His liver regenerated overnight, thus providing the eagle with
eternal food and Prometheus with eternal torture [16].
However, liver regeneration cannot always be relied upon to take place after liver
injury. In the event of ineffective or even total absence of liver regeneration, the
life-threatening picture of liver failure may appear [4]. In other cases there may be
incomplete liver regeneration, a condition known as hepatic fibrosis, in which the
damaged liver tissue is replaced not by cells of the same kind, but by substitute
tissue. This state of hepatic fibrosis tends to progress, ultimately leading to the
destruction of the liver architecture, terminating in hepatic cirrhosis with the
clinical picture of chronic liver failure, a condition with a notoriously bad prognosis
[6].
Despite vigorous research efforts during the last few decades, the pathophysiology
of liver regeneration is still largely unknown. In view of the growing importance of
liver regeneration as the basis for treatment of many liver diseases, one of the
objectives of current research is to achieve a better understanding of the
extracellular and intracellular signals which govern liver regeneration, and thereby
to develop new therapeutic strategies for stimulation of liver regeneration and
prevention of liver fibrosis, in the hope of reducing the incidence and morbidity of
hepatic cirrhosis.
Etiology of canine hepatitis
An infectious etiology is often suspected in canine hepatitis, but despite the
existence of many known etiologic factors, the etiology of individual causes stays
largely unknown [2].
Many infectious agents known for causing hepatitis have yet been identified;
viruses (canine adenovirus/Rubarth virus), bacteria (Clostridium piliformis,
Leptospira), protozoa (Toxoplasma gondii, Neospora, Leishmania) and fungi
(Histoplasma capsulatum). Several toxic agents are also known for causing
hepatitis; copper, blue green algae and therapeutic drugs such as trimetoprim
sulfa, benzodiazepine and carprofen [6]. But as said, the etiology of individual
causes stays largely unknown.
Histological changes in canine hepatitis
Hepatitis in veterinary hepatology always includes hepatocellular cell death;
apoptosis and/or necrosis, and an inflammatory infiltrate varying in type and
extend [12]. Furthermore, acute hepatitis (AH) sometimes features regeneration,
whereas chronic hepatitis (CH) always conveys regeneration and fibrosis. Lobular
dissecting hepatitis (LDH) is a special form of chronic hepatitis, in which the
lobular architecture is completely disrupted by fine fibrotic septa [6].
Following hepatic inflammation, the liver gives rise to a tissue repair response in
order to regain its physiological function.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Liver tissue repair
This tissue repair respons (hepatocellular regeneration) can occur by two means;
A) replication of mature hepatocytes and cholangiocytes and,
B) in case of hampered hepatocytic reproduction, also by proliferation of the
liver specific stem cell, the liver progenitor cell (LPC) [8].
As said, the progenitor cells are activated when hepatocellular proliferative
capacity falls short. They ultimately differentiate into new hepatocytes or biliary
epithelial cells (Fig. 1) [8].
Hepatobiliary
cells
Hepatocytes
Cholangiocytes
Reactive ductuli
Hepatic progenitor
cells
Fig. 1; Differentiation pathways of the LPC [8]
As a general rule, replication of existing hepatocytes is the quickest and most
efficient way to generate hepatocytes for liver regeneration and repair. LPC’s
usually replicate and differentiate into hepatocytes or cholangiocytes only when
the replication of mature cells is impaired or entirely blocked. Hepatocytes,
cholangiocytes and LPC’s all originate from endodermal-derived hepatoblasts (Fig.
2) during embryonic development [5].
Fig. 2; Cell lineages in the liver [5]
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
In adult livers, both hepatocytes and cholangiocytes can replicate. LPC’s form a
bipotential reserve compartment capable of generating hepatocytes and
cholangiocytes whenever regular replication is inadequate [5]. It is thought that
cells surrounding the progenitor cells, amongst which the mesenchymal cells,
release signals that regulate the activity of the progenitor cells. These surrounding
cells with their signals and signal transduction pathways are called the progenitor
cell niche.
These signal transduction pathways involved in adult progenitor cell activation
have yet been described in other organs with a continuous cellular turnover, for
example the intestines. The homeostatic self-renewal of the intestine depends on a
complex interplay between processes involved in cell proliferation, differentiation,
migration, adhesion and cell death. This cellular response is coordinated by a
relatively small number of highly evolutionarily conserved signaling pathways,
which include the Notch and Wnt signaling pathways [3].
Since development of the liver from the ventral foregut endoderm is also
influenced by Notch and Wnt signalling, these signalling pathways may also be
involved in the regulation and control of LPC’s in the adult liver later in life [17].
Signalling pathways
Signal transduction pathways are efficient systems that allow cells to amplify faint
signals in order to create intense, vital responses. A key element of virtually any
signaling pathway is the interaction and regulation of various intracellular
intermediates within the pathway.
The Notch signalling pathway (Fig. 3)
Notch signalling is an evolutionary conserved
mechanism, used to regulate cell fate decisions
during the development of many cell lineages.
Members of the Notch receptor family encode large
transmembrane
proteins
that
mediate
communication between neighbouring cells during
cell-cell contact, by binding to ligands expressed on
adjacent cells. Ligand-mediated activation of Notch
induces the proteolytic release and nuclear
translocation of the Notch intracellular domain. This
activated intracellular domain of Notch receptors
interacts with DNA binding proteins to directly
modulate the expression of target genes. Therefore,
Notch mediates direct signal transduction from the
cell surface to the nucleus, allowing cells to directly
regulate gene expression in neighbouring cells and
influence their development [3]
Fig. 3; Notch signalling pathway
[Wikipedia]
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
The Jagged family of transmembrane proteins are ligands for Notch receptors, which
control the proliferation and/or differentiation of many cell lineages.
In recent human liver tissue studies [11] expression of Jagged-1 was seen in normal as
well as in diseased liver tissue. Interestingly, Jagged-1 expression was significantly
up-regulated in diseased liver tissue (Fig. 4). This suggests that the Notch signaling
pathway may play a role in Human liver regeneration observed during liver disease.
Since little is known about the involvement of the Notch signaling pathway in LPC
activation in the dog, it makes this an interesting subject for research.
A
B
Fig. 4; Immunolocalization of Jagged-1 expression in normal and diseased adult human liver tissue. A;
In the parenchyma of normal liver tissue, Jagged-1 is localized to the cell membrane of hepatocytes
(arrowheads) and the endothelium of the hepatic vein (arrow). B; Strong Jagged-1 immunoreactivity is
associated with reactive bile ductules in PBC liver tissue (black arrows) [12].
The Wnt signalling pathway (Fig. 5)
The Wnt signalling pathway is highly conserved
throughout animal development where it exerts
pleiotropic effects on cell proliferation, differentiation
and polarity or migration. The Wnt signalling pathway is
identified as a signalling pathway that critically
regulates various postnatal stem cell compartments,
including the hematopoietic, skin and enteric systems.
The central mediator of canonical Wnt signalling is ßcatenin, which acts as an activator of gene transcription
by binding to the nuclear complex.
During liver development, ß-catenin was shown to
critically regulate hepatic progenitor cell proliferation.
It was even shown that over expression or inhibition of
ß-catenin during development results in either an
increase or a decrease in overall liver size, respectively
[17].
Fig. 5; Wnt signalling pathway [11]
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
So recent evidence indicates that ß-catenin is crucial for cell division during
embryonic liver development and liver regeneration. More recently, ß-catenin is also
shown to regulate the proliferative response of hepatic LPC’s in human hepatocellular
carcinoma tissue(Fig. 6) [17].
Fig. 6; Expression of ß-catenin in OV6+ (a marker for LPC’s) cells in human liver diseases. A; in normal
liver, OV6 are mainly expressed in bile ducts and periportal hepatocytes. B; in liver cirrhosis, OV6
expression are observed as small single cells in the periportal area or as reactive ductular structures at
the portal-parenchymal interface. C and D; serial sections of HCC in a patient were stained with antiOV6 and anti– ß-catenin. OV6 expression was observed in a subset of intermediate hepatocyte-like cells
in hepatocellular carcinoma tissue (C). Nuclear staining of ß-catenin was higher in the OV6+
hepatocellular carcinoma cells (C) than the adjacent OV6 hepatocellular carcinoma cells (D) [18].
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Summary of previous research
Previous study on these signalling pathways has been done by B.A. Schotanus et al., in
which healthy liver tissue was compared to a specific type of chronic liver injury;
lobular dissecting hepatitis (LDH), by use of laser-microdissection, gene expression
studies (Q-PCR), and immunohistochemistry/-fluorescence.
Results showed an activated state of the Notch and Wnt pathways in the activated
LPC (niches). The Wnt pathway seemed to be particularly involved in proliferation of
the LPC, whereas the Notch pathway seemed to be particularly involved in LPC
differentiation into biliary cells and seemed to function inhibitory on LPC
differentiation into hepatocytes.
Aim of the research project
My aim for this research is to study the liver progenitor cells and the mechanisms of
their activation in the cell niche, by use of immunohistochemical techniques. Thereby
concentrating specifically on the Wnt and Notch signalling pathways involved in LPC
activation. This will be done in liver diseases with different stages of inflammation,
fibrosis and pathophysiology. This will be compared to previously collected data from
healthy livers and LDH. This will help us to better understand the pathophysiology of
liver regeneration in different diseases.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Materials and methods
Immunohistochemistry
Immunohistochemistry (or IHC) is a method for demonstrating the presence and
location of proteins in tissue sections so it enables the observation of processes in the
context of (intact) tissue. Immunohistochemical staining is accomplished with
antibodies that recognize the target protein. Since antibodies are highly specific, the
antibody will bind only to the protein of interest in the tissue section (Fig. 7). The
antibody-antigen interaction is visualized using chromogenic detection, in which an
enzyme conjugated to the antibody cleaves a substrate to produce a colored
precipitate at the location of the protein. For example; a Notch1 specific antibody to
stain the Notch signaling pathway and a β-catenin specific antibody to stain the Wnt
signalling pathway were used. When using a new antibody in IHC, the antibody must
be tested to find the optimal staining conditions.
Chromogen
secondary antibody
primary antibody
Fig. 7; Immunohistochemical binding of specific antibodies [10]
Paraffin embedded tissue versus frozen tissue material
Immunohistochemistry can be carried out on paraffin-embeded liver tissue as well as
on frozen liver tissue. Initially, the goal was to stain Notch1 and β-catenin in paraffinembedded liver tissue. For this there are a couple of reasons which are listed below.
Advantages of paraffin embedded material versus frozen material [10];
-greater availability of paraffin-embedded, diseased liver tissue,
-better morphology of paraffin-embedded material,
-better resolution at higher magnifications,
-better cutting possibilities (less cutting-artifacts),
-frozen sections subjected to immunohistochemical procedures often show
deleterious morphological changes, including chromatolysis and apparent loss of
membranes.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Disadvantages of paraffin embedded material versus frozen material [10];
-fixation and embedding cause antigen masking. In order to overcome the
drawback of antigen loss, enzymatic- or heat- mediated antigen retrieval has to be
used,
-cryostat sections give much better antigen preservation than paraffin sections.
If immunohistochemical staining on paraffin-fixed liver tissue would prove unsatisfying
there was also frozen liver tissue available to obtain data from.
Immunohistochemistry on paraffin-embedded liver tissue
Single immunostaining was performed on formalin fixed paraffin embedded tissue
samples for Notch-1 (ab sc-6014 from Santa Cruz Biotechnology, rabbit polyclonal)
and β-catenin (ab 2982 from Abcam, rabbit polyclonal).
To optimize staining results, different antigen retrievals, boiling times, H2O2 blocks,
washes, serum blocks and antibody dilutions were tried for Notch-1 (see attachment A
pa 24) as well as for β-catenin (see attachment B page 28). For the protocols used see
attachment C on page 30 for Notch-1 and attachment D on page 31 for β-catenin.
Since outcome of the different optimization protocols was not satisfying, which is
shown in earlier mentioned attachments, Notch-1 and β-catenin staining was
repeated on frozen tissue samples.
Immunohistochemistry on frozen liver tissue
Frozen liver tissue of different types of liver diseases were stained for CK 7, Notch
and β-catenin. CK 7 staining was performed mainly to determine localisation and the
amount of progenitor cells present in the slides.
Cryosections (6 µm) were cut on a cryostat at -20°C. The collected slides were dried
at room temperature for three hours and fixated in acetone for ten minutes and dried
at air for ten minutes again. Sections were then stored in aluminum foil at – 70 and
dried at air for 20 min. in aluminum foil and dried at air for 10 min. without
aluminum foil before use. For CK7, brief washing steps were performed in PBS or PBS
0.1% Tween20. Endogenous peroxidise activity was blocked in RTU DAKO standard
H2O2, background staining was blocked with normal goat serum (1:10 in PBS), antibody
incubations (with Mouse anti CK7 1:50) occurred at RT for 30 min. and visualization
was obtained by diaminobenzidine and counter stain consisted of a 10 sec.
haematoxylin stain. Slides were covered with Vectamount. For the complete staining
protocol see attachment E, page 32.
For Notch-1 and ß-catenin, brief washing steps were performed in TBS or TBS-0.025%
Triton. Endogenous peroxidise activity was blocked in RTU DAKO standard H2O2,
background staining was blocked with normal goat serum (1:10 in TBS), antibody
incubations (1:50 for Notch-1 and 1:200 for ß-catenin in TBS-0.025% Triton) occurred
overnight and visualization was obtained by diaminobenzidine and counter stain
consisted of a 10 sec. haematoxylin stain. Slides were covered with Vectamount.
For the complete staining protocol see attachment F and G on page 33 and 34.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Liver tissue samples used
Different types of frozen liver tissue were used for staining, namely;
-normal liver tissue;
n=6
-acute hepatitis;
n=3
-active cirrhosis;
n=8
-primary portal vein hypoplasia
n=5
-extra hepatic bile duct obstruction
n=2
-negative control (liver tissue)
n=1
-positive control (intestine)
n=1
The number of tissue samples used per disease depended on the availability.
The healthy liver tissue samples used concerned surplus-liver tissue form dogs used in
non-liver related experiments.
The diseased liver tissue samples were obtained from patients submitted to the Clinic
of Companion Animals of Utrecht University. The project was approved by the
responsible ethical committees according to the Dutch legislation.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Results
Paraffin-embedded liver tissue
As mentioned in the materials and methods, different antigen retrievals, washings,
blockings etc. were used in the staining protocol to optimize staining results for
Notch-1 as well as for ß-catenin on paraffin-embedded liver tissue.
Unfortunately, none of the variables used in the protocol resulted in satisfying
staining results, which varied from no staining at all to such high background staining
that no conclusions could be drawn from the stainings. A couple of examples of
staining results are shown below to demonstrate different, unsatisfying, outcomes.
An overview of all the different staining results can be found at attachment A and B,
page 24 and 28.
Fig. 8; Staining result after using Citrate (pH 6.0) as antigen retrieval (boiled for 30 min. in a
microwave), a 0.35% H202 block, PBS(T) as washing, 10% normal goat serum for serum blocking and a
Notch 1 antibody dilution of 1:25. Results show that there is only little staining of cholangiocytes and
the liver parenchyma.
Fig.9; Staining result after using TRIS EDTA (pH 10.0) as antigen retrieval (boiled for 30 min. in a
microwave), a 0.35% H202 block, PBS(T) as washing, 10% normal goat serum for serum blocking and a
Notch 1 antibody dilution of 1:25. Results show that there is bile duct staining, periportal and –venous
field staining but such high background staining that no conclusions can be drawn (see inset).
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Fig.10; Staining results after using Sodium citrate (pH 6.0) as antigen retrieval (boiled for 15 min. in a
microwave), a 0.35% H202 block, 0.025% T TBS as washing, 3% BSA in T TBS for serum blocking and a ßcatenin antibody dilution of 1:50. Results show that there is little staining of the cholangiocytes,
staining of the hepatocytes and no staining of the periportal area. Staining wasn’t specific enough to
draw conclusions.
Fig. 11; Staining results after using a standard antigen unmasking solution as antigen retrieval (boiled
for 3 min. in a microwave), a 0.35% H202 block, 0.025% T TBS as washing, 10% normal goat serum for
serum blocking and a ß-catenin antibody dilution of 1:50. Results show that there is a lot of background
staining but no specific staining.
Since no conclusions could be drawn concerning the involvement of β-catenin and
Notch-1 in LPC activation on paraffin-embedded liver tissue, staining was performed
on frozen liver tissue.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Frozen liver tissue
Healthy liver tissue
In healthy liver tissue only few CK 7 positive cells are present (Fig. 12A). Notch-1
staining shows that staining is mainly concentrated in the hepatocytes, which
moderately stain membranously. Endothelium also stains moderately. Cholangiocytes
stain weak. There is no staining of CK 7 positive cells (Fig. 12B). β-catenin staining
mainly shows moderate staining of hepatocytes (membranous) and cholangiocytes.
Areas of CK 7 positive cells (Fig. 13A) also show strong β-catenin staining
(membranous) (Fig. 13B).
A
B
Fig. 12; A; CK 7 staining on healthy liver tissue, B; Notch staining on healthy liver tissue.
A
B
Fig. 13; A; CK 7 staining on healthy liver tissue, B; β-catenin staining on healthy liver tissue.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Acute Hepatitis
In acute hepatitis the overall amount of CK 7 positive cells is slightly increased (then
called reactive ductules (Fig. 14A)). Notch staining mainly shows little hepatocytes
(membranous and cytoplasmatic) and endothelium staining. There is none (two out of
three slides) to little (one out of three slides(Fig. 14B)) staining of CK 7 positive cells.
β-catenin staining mainly shows moderate staining of hepatocytes (membranous) and
cholangiocytes. In most slides stained for β-catenin, areas of CK 7 positive cells
showed increased staining for β-catenin (Fig. 15).
A
B
Fig. 14; A; CK 7 staining on acute hepatitis liver tissue, B; Notch staining on acute hepatitis liver
tissue.
Fig. 15; β-catenin staining on acute hepatitis liver tissue
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Active cirrhosis
In seven out of eight slides of cirrhotic liver tissue a strong increase of CK 7 positive
cells (reactive ductules) is seen, mainly in the fibrotic septae (Fig. 16A). In the Notch
staining, hepatocytes mainly stain membranous and in four out of eight slides
cholangiocytes stain weak to little. Two slides show no staining, four slides show little
to weak staining, and two slides show moderate to strong Notch staining (white
arrows) of active ductules. In the β-catenin staining, hepatocytes stain mainly
cytoplasmatic, three slides also stain membranous. Three out of eight slides show
little, and five out of eight slides show a strong staining of reactive ductules (Fig. 17).
A
B
Fig. 16; A; CK 7 staining on cirrhotic liver tissue, B; Notch staining on cirrhotic liver tissue. The white
arrows show areas of CK 7 as well as Notch positivity. The greater magnifications show clear positive
staining for CK 7 as well as for Notch.
A
B
Fig. 17; A; CK 7 staining on cirrhotic liver tissue, B; β-catenin staining on cirrhotic liver tissue. The
greater magnifications show clear positive staining for CK 7 as well as for β-catenin.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Primary portal vein hypoplasia (PPVH)
In the PPVH slides two out of five slides show little, three show moderate and one
slide shows strong increase in CK 7 positive cells, mainly in areas with increased
amounts of fibrosis, which is typically for this type of liver disease (Fig. 18A). Notch
staining shows little staining of hepatocytes (membranous as well as cytoplasmatic)
and virtually no staining of cholangiocytes. Except for one slide in which moderate
staining can be seen in the area of CK 7 positive cells (a slide with increased amounts
of CK 7 positive cells (Fig. 18B)), no staining was observed of CK 7 positive cells. In
the β-catenin staining, hepatocytes showed cytoplasmatic as well as membranous
staining. Cholangiocytes, when present (three out of five slides), showed little
staining. A strong staining of CK 7 positive cells was seen in three out of five slides
(Fig. 19). In these slides an increase in CK 7 positive cells was observed as well.
A
B
Fig. 18; A; CK 7 staining on primary portal vein hypoplasia liver tissue, B; Notch staining on primary
portal vein hypoplasia liver tissue.
Fig. 19; β-catenin staining on primary portal vein hypoplasia liver tissue.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Extra hepatic bile duct obstruction
In liver slides with extra hepatic bile duct obstruction a minimal increase in CK 7
positive cells can be seen (Fig. 20A). There is no specific staining of these cells or
areas in the Notch-1 stained slides (Fig. 20B). Cholangiocytes stain negative and
hepatocytes mainly stain membranous. In the slides stained for β-catenin, little
staining of CK 7 positive cells was observed (Fig. 21). Cholangiocytes stain moderate,
hepatocytes mainly stain membranous.
A
B
Fig. 20; A; CK 7 staining on extra hepatic bile duct obstruction liver tissue, B; Notch staining on extra
hepatic bile duct obstruction liver tissue.
Fig. 21; β-catenin staining on extra hepatic bile duct obstruction liver tissue.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Negative control (liver tissue)
In the healthy liver tissue slides used for negative control, staining can be seen in the
slides stained for Notch as well as in the slides stained for β-catenin. This is mainly
background staining and not specific (Fig. 22A and 22B).
A
B
Fig. 22; A; Notch staining (negative control) on healthy liver tissue, B; β-catenin staining (negative
control) on healthy liver tissue.
Positive control (intestine)
In the intestinal tissue slides used for positive control, strong positive staining is
mainly seen, as expected [3], in the crypts of Lieberkühn. This applies to the Notch
staining (Fig. 23A) as well as for the β-catenin staining (Fig. 23B).
A
B
Fig. 23; A; Notch staining (positive control) on intestinal tissue, B; β-catenin staining (positive control)
on intestinal tissue.
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-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Discussion
Paraffin-embedded liver tissue
Although many attempts were done to optimize staining results on paraffin-embedded
liver tissue for Notch-1 as well as for β-catenin, unfortunately none of them proved
satisfying enough to be able to draw conclusions out of the staining results. There are
many possibilities for why staining results come out unsatisfying, of which a couple
are listed below;
-The antibody may not be suitable for IHC procedures which reveal the protein in its
native. This is not the case, since staining on frozen liver tissue showed that the
antibody was suitable.
-The protein of interest is not abundantly present in the tissue. Staining on frozen
liver tissue showed that this is not the reason because our antigen of interest was
abundantly present.
-Fixation procedures (using formalin and paraformaldehyde fixatives) may be
modifying the epitope the antibody recognizes.
-The secondary antibody may be binding non-specifically (damaged),
-Some antibodies only work on fresh, unfixed, frozen tissue,
- Because antigen-antibody reactions are reversible, the simple immune complexes
formed on the tissue may dissociate during the washing cycles used in
immunohistochemistry. The ease and degree of dissociation vary from antibody to
antibody.
Unfortunately, time was short to continue optimizing staining results. Hopefully in the
future the right staining protocol will be found so the advantages of the use of
paraffin-embedded tissue can be exploited.
Frozen liver tissue
Liver regeneration is a very complex process influenced by a great variety of growth
factors, cytokines, cell-cell interactions and the liver specific progenitor cell (LPC). In
this study the involvement of the signalling pathways Notch and Wnt in the activation
of LPC’s was investigated by the use of immunohistochemistry. In the past, research
has already suggested that both Notch and Wnt play an important role in the
activation of LPC’s in human and rat livers [1] [8].
Research done by Schotanus et al. [15] demonstrated the role of Wnt and Notch in
proliferation and differentiation of the LPC’s during a specific type of liver disease in
the dog; lobular dissecting hepatitis (LDH). LDH is a type of liver disease which is
characterised by extended fibrosis throughout the liver tissue. In this severe state of
liver tissue damage replication of mature hepatocytes isn’t sufficient to regain its
physiological function. Therefore LPC’s replicate (their numbers increase) and
differentiate into hepatocytes and cholangiocytes which was also observed in the CK 7
staining. Notch-1 and β-catenin were up regulated in the LDH tissue compared to
healthy liver tissue, demonstrating the involvement of signalling pathways Notch-1
and Wnt in the activation of LPC’s in this type of liver disease. Moreover, it was
suggested that Wnt was especially involved in proliferation of the LPC’s and their
differentiation into hepatocytes, whereas Notch-1 was suggested to be especially
involved in differentiation of the LPC’s into cholangiocytes [15].
This prompted us to study LPC’s and their signalling pathways in different types of
liver disease as well and compare them to previously collected data.
In Healthy liver tissue small amounts of LPC’s were observed. These cells stained
negative for Notch-1 and showed moderate membranous staining for β-catenin,
21
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
showing that Notch-1 and Wnt aren’t activated in resting LPC’s. This suits results of
previous research performed on healthy liver tissue of rats [1] [8].
In acute hepatitis, a disease which in general is mainly characterised by extensive
inflammation, a few reactive ductules were seen, suggesting a minimal activation of
LPC’s. This could indicate that the replication of mature hepatocytes and
cholangiocytes, up to a certain level, is sufficient enough for liver regeneration. Since
reactive ductules show increased membranous staining for β-catenin and no Notch-1
staining, this suggests that there is indeed proliferation of LPC’s and differentiation
into hepatocytes, but no differentiation into cholangiocytes (yet). The one slide in
which Notch-1 staining of reactive ductules was seen (Fig. 14A and B), indicates that
in this case LPC’s did differentiate into cholangiocytes as well. As acute hepatitis
becomes more chronic and tissue damage becomes more severe, proliferation and
differentiation can increase, as can be seen in LDH and active cirrhosis.
In active cirrhosis, liver injury is merely characterised by an extensive increase in
fibrotic tissue and less inflammation. A large amount of reactive ductules were seen,
showing that in this severe state of liver tissue damage replication of mature
hepatocytes and cholangiocytes falls short and LPC’s come into play in order of liver
regeneration. Notch-1 and β-catenin staining both showed an overall increase in
staining intensity of the reactive ductules. Based on the suggestion that Wnt is merely
involved in proliferation while notch in differentiation, this suggests that under these
circumstances there is an increased demand for cholangiocytes and hepatocytes and
therefore both Notch-1 and β-catenin show increased staining. This suits earlier
staining results of LDH tissue in which increased staining of β-catenin and Notch-1 was
seen as well [15].
In PPVH, a disease in which extended fibrosis can be found in the portal area without
an inflammatory component, little, moderate and high amounts of reactive ductules
were seen. Since the exact pathophysiology of PPVH is still not clear, the variation in
reactive ductules amounts could be due to (not yet known) variation in severity or
expression of PPVH. Notch-1 staining showed only little positivity in one slide, in
area’s where large amounts of reactive ductules were found as well. Other slides with
increased numbers of reactive ductules didn’t show Notch-1 positivity. This suggests
that in some forms of PPVH, LPC’s differentiate into cholangiocytes and in other
forms they don’t. The exact reason for this is not yet clear. The β-catenin staining of
PPVH tissue showed increased positivity of reactive ductules in all three slides in
which the amount of reactive ductules was increased. In the two slides in which no
increase of reactive ductules was seen, only little β-catenin positivity was seen. This
shows that in certain forms of PPVH, LPC’s proliferate and differentiate into
hepatocytes, influenced by Wnt.
In extra hepatic bile duct obstruction, a disease which is primarily a disease of the
biliary tract, Notch-1 stained negative and β-catenin stained little in one slide. Since
β-catenin staining showed little positivity in the slide in which reactive ductules were
seen as well, this suggests that in this specific slide the LPC’s were needed to
proliferate and differentiate into hepatocytes. Unfortunately only two tissue samples
of extra hepatic bile duct obstruction were available, making it difficult to determine
the exact role of the LPC’s and their activation mechanisms in extra hepatic bile duct
obstruction.
22
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Since negative controls for Notch-1 as well as for β-catenin showed positive staining,
suggestions and conclusions based on the stainings can’t be made with certainty.
Reason for the negative control slides to be positive could be a lack of specificity of
the antibody, although the rest of the results don’t seem to support this option
because of the staining intention variation observed in other slides. Another reason
could be that errors have occurred during the processing of the slides. We weren’t
able to identify the exact reason for the negative control slides to be positive. In the
future, staining results will hopefully be accompanied by negative controls, making
results more reliable.
Taken together, the obtained data provide insight in the role of signaling pathways
Notch-1 and Wnt in the activation of LPC’s in different liver diseases in the dog.
Although the precise role of these signaling pathways has not been demonstrated
unequivocally in the current report, results form a suitable base for future research.
Current research
Current research on Notch and Wnt is performed by Alfonso Broers. With the use of
specific cell-lines (BDE, HepaRG and THLE5b) and interventions (ligands-Inhibitors and
siRNA), he hopefully will be able to give more insight in the role of Notch and Wnt in
LPC activation.
Acknowledgment
I would like to thank my supervisor Baukje Schotanus, Louis Penning, Bas Brinkhof,
Adri Slob, Jeannette Wolfswinkel and all my other colleagues and fellow students at
the laboratory for their intellectual and technical support at all stages throughout the
study.
23
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Apte, A. et al., Wnt/Beta-Catenin Signaling Mediates Oval Cell Response in
Rodents. Hepatology, Vol. 47, No. 1, 2008.
Boomkens, S.Y., et al., Hepatitis with special reference to dogs. A review on
the pathogenesis and infectious etiologies, including unpublished results of
recent own studies. Vet Q, 2004. 26(3): p. 107-14.
Es, van, J.H. and H. Clevers, Notch and Wnt inhibitors as potential new drugs
for intestinal neoplastic disease. Trends Mol Med, 2005. 11(11): p. 496-502.
Fausto, N., Hepatocyte differentiation and liver progenitor cells. Curr Opin
Cell Biol, 1990. 2(6): p. 1036-42.
Fausto, N., Liver regeneration and repair: hepatocytes, progenitor cells, and
stem cells. Hepatology, 2004. 39(6): p. 1477-87.
Fre, S. et al., The role of Notch receptor expression in bile duct development
and disease. Nature, Vol 435|16 June 2005|doi:10.1038/nature03589.
Ijzer, J., Liver fibrosis and regeneration in dogs and cats: An
immunohistochemical approach. 2008, Utrecht. 159.
Jensen, C.H. et al., Transit-Amplifying Ductular (Oval) Cells and Their
Hepatocytic Progeny Are Characterized by a Novel and Distinctive Expression
of Delta-Like Protein/Preadipocyte Factor 1/Fetal Antigen 1. American Journal
of Pathology, Vol. 164, No. 4, April 2004.
Libbrecht, L. and T. Roskams, Hepatic progenitor cells in human liver diseases.
Semin Cell Dev Biol, 2002. 13(6): p. 389-96.
Marc Key, P.D., Karen Atwood, B.S. MT (ASCP) CLS, Kirsten Bisgaard, B.S. et
al., Immunohistochemical Staining Methods Fourth Edition. 2006: Dako int.
174.
Moon, R.T., et al., WNT and beta-catenin signalling: diseases and therapies.
Nat Rev Genet, 2004. 5(9): p. 691-701.
Nijjar, S.S., et al., Altered Notch ligand expression in human liver disease:
further evidence for a role of the Notch signaling pathway in hepatic
neovascularization and biliary ductular defects. Am J Pathol, 2002. 160(5): p.
1695-703.
Rothuizen, J., Bunch, SE, Charles, JA, CUllen, JM, Desmet, V, Szatmari, V, et
al, WSAVA standards for clinical and histological diagnosis of canine and feline
liver diseases. 2006, Edinburgh: Saunders Elsevier.
Scheving, L.A. and W.E. Russell, Beta-catenin in the liver: an integrator of
proliferation and metabolism? Gastroenterology, 2006. 131(5): p. 1641-3.
Schotanus et al., Increased Wnt and Notch signaling in activated liver
progenitor cell niches (submitted for publication).
Tygstrup, N. and H.C. Bisgaard, [Liver regeneration. The Prometheus myth in
the light of molecular biology]. Ugeskr Laeger, 1998. 160(52): p. 7612-5.
Yang, W., et al., Wnt/beta-catenin signaling contributes to activation of
normal and tumorigenic liver progenitor cells. Cancer Res, 2008. 68(11): p.
4287-95.
Zaret, K.S., Molecular genetics of early liver development. Annu Rev Physiol,
1996. 58: p. 231-51.
24
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Attachment A
Paraffin-embedded liver tissue, Notch 1 protocol- and results table
COUPE
ANTIGEN
RETRIEVAL
N50
None
N150
None
(BOILING)
TIME
H2O2
BLOCK
WASH
-
0.35%
PBS(T)
-
0.35%
PBS(T)
C50
Citrate pH
6,0
15 min
0.35%
PBS(T)
C150
Citrate pH
6,0
15 min
0.35%
PBS(T)
P50
Pepsin 0,4%
15 min
0.35%
PBS(T)
P150
Pepsin 0,4%
15 min
0.35%
PBS(T)
P25
Proteinase K
15 min
0.35%
PBS(T)
P500
Proteinase K
15 min
0.35%
PBS(T)
C25
Citrate pH
6,0
30 min
0.35%
PBS(T)
C500
Citrate pH
6,0
30 min
0.35%
PBS(T)
M25
MiliQ
30 min
0.35%
PBS(T)
M500
MiliQ
30 min
0.35%
PBS(T)
SERUM
BLOCK
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
E25A
TRIS EDTA pH
9,0
30 min
0.35%
PBS(T)
E500
TRIS EDTA pH
9,0
30 min
0.35%
PBS(T)
10%
normal
goat
10%
normal
goat
PBS(T)
10%
normal
goat
--
Trypsin 0,4%
15 min
0.35%
25
ANTIBODY
FINDINGS
Negative
1:50
Negative
1:150
1:50
Little bile duct
staining No
membrane staining
Negative
1:150
Negative
1:50
Negative
1:150
Negative
1:25
Negative
1:500
Negative
1:25
Negative
1:500
1:25
Little bile duct
staining No
membrane staining
No background
staining
Negative
1:500
1:25
Little bile duct
staining Little/no
membrane staining
Negative
1:500
Slide let loose
1:25
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
--
Trypsin 0,4%
15 min
0.35%
PBS(T)
E25B
TRIS EDTA pH
10,0
LDH;
TRIS EDTA pH
9,0
30 min
0.35%
PBS(T)
AUS50A
AUS
3 min
0.35%
PBS(T)
E30T50
TRIS EDTA pH
10,0
E10T50
TRIS EDTA pH
10,0
E30T10
0
TRIS EDTA pH
10,0
E10T10
0
TRIS EDTA pH
10,0
T10 50
EGTA pH
10,0
T30 50
EGTA pH
10,0
ABGS
10 50
TRIS EDTA pH
10,0
ABGS
30 50
TRIS EDTA pH
10,0
SH2O2
10 50
TRIS EDTA pH
10,0
30 min
30 min
10 min
30 min
10 min
10 min
30 min
10 min
30 min
10 min
0.35%
0.35%
0.35%
0.35%
0.35%
0.35%
0.35%
0.35%
0.35%
standard
PBS(T)
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
Slide let loose
1:50
1:25
1:50
Negative
1:50
(T)TBS
10%
normal
goat
1:50
(T)TBS
10%
normal
goat
1:50
(T)TBS
10%
normal
goat
1:100
(T)TBS
10%
normal
goat
1:100
PBS(T)
10%
normal
goat
1:50
PBS(T)
10%
normal
goat
1:50
PBS(T)
10%
normal
goat
In 10% goat
serum 1:25
PBS(T)
10%
normal
goat
In 10% goat
serum 1:25
PBS(T)
10%
normal
goat
26
Bile duct staining
Little membrane
staining High
background staining
Periportal & -venous
field staining
Bile duct staining
Little membrane
staining High
background staining
High background
staining No
membrane staining
Periportal & -venous
field staining
High background
staining No
membrane staining
Periportal & -venous
field staining
High background
staining No
membrane staining
Periportal & -venous
field staining
High background
staining No
membrane staining
Periportal & -venous
field staining
High background
staining No
membrane staining
Periportal & -venous
field staining
High background
staining No
membrane staining
Periportal & -venous
field staining
Less background
staining No
membrane staining
Periportal & -venous
field staining
Negative
Negative
1:50
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
SH2O2
30 50
GS10
50
TRIS EDTA pH
10,0
TRIS EDTA pH
10,0
30 min
10 min
standard
0.35%
PBS(T)
10%
normal
goat
PBS(T)
20%
normal
goat
GS30
50
TRIS EDTA pH
10,0
30 min
0.35%
PBS(T)
30 GS
50
TRIS EDTA pH
10,0
30 min
0.35%
PBS(T)
AB
20GS50
20%
normal
goat
30%
normal
goat
10%
normal
goat
BSA 50
TRIS EDTA pH
10,0
TRIS EDTA pH
10,0
SH2O2
50
TRIS EDTA pH
10,0
LDH 50
TRIS EDTA pH
10,0
30 min
standard
PBS(T)
GGS 50
TRIS EDTA pH
10,0
30 min
standard
PBS(T)
3% BSA
30%
normal
goat
30%
normal
goat
10%
normal
goat
TRIS EDTA pH
10,0
30 min
standard
PBS(T)
BSA 1%
BSAB2
50
TRIS EDTA pH
10,0
30 min
standard
PBS(T)
T30A
TRIS EDTA pH
10,0
30 min
standard
PBS(T)
T20A
TRIS EDTA pH
10,0
20 min
standard
PBS(T)
T30B
TRIS EDTA pH
10,0
30 min
standard
PBS(T)
T20B
TRIS EDTA pH
10,0
20 min
standard
PBS(T)
T30C
TRIS EDTA pH
10,0
30 min
standard
PBS(T)
T20C
TW 35
A
TRIS EDTA pH
10,0
TRIS EDTA pH
10,0
20 min
30 min
waterbath
standard
0.35%
After
A.R.
PBS(T)
BSAB1
50
30 min
0.35%
PBS(T)
30 min
0.35%
PBS(T)
30 min
standard
PBS(T)
PBS(T)
27
BSA
0,05%
15%
normal
goat
15%
normal
goat
15%
normal
goat
15%
normal
goat
15%
normal
goat
15%
normal
goat
10%
normal
goat
1:50
1:50
1:50
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
Negative
1:50
In 20% goat
serum 1:50
1:50
High background
staining No
membrane staining
Periportal & -venous
field staining
Negative
Negative
1:50
In 10% goat
serum 1:50
In 10% goat
serum 1:50
In 1% BSA
1:50
In 0,01%
BSA 1:50
In 15% goat
serum 1:50
1night
In 15% goat
serum 1:50
1night
In 15% goat
serum 1:50
2nights
In 15% goat
serum 1:50
2nights
In 15% goat
serum 1:50
day
In 15% goat
serum 1:50
day
1:25
Little background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
Less background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRATW S A
TRIS EDTA pH
10,0
30 min
waterbath
TM 35
A
TRIS EDTA pH
10,0
30 min
microwave
TM S A
TRIS EDTA pH
10,0
30 min
microwave
TW 35
B
TRIS EDTA pH
10,0
30 min
waterbath
TW S B
TM 35
B
TM S B
TRIS EDTA pH
10,0
TRIS EDTA pH
10,0
TRIS EDTA pH
10,0
30 min
waterbath
30 min
microwave
30 min
microwave
Standard
After
A.R.
0.35%
After
A.R.
Standard
After
A.R.
0.35%
Before
A.R.
Standard
Before
A.R.
0.35%
Before
A.R.
Standard
Before
A.R.
PBS(T)
PBS(T)
PBS(T)
PBS(T)
PBS(T)
PBS(T)
PBS(T)
28
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
10%
normal
goat
1:25
1:25
1:25
1:25
1:25
1:25
1:25
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
High background
staining No
membrane staining
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Attachment B
Paraffin-embedded liver tissue: β-catenin protocol- and results table
ANTIGEN
RETRIEVAL
BOILING
TIME
H2O2
BLOCK
WASH
3 min
0.35%
0.025%
T TBS
C50
None
Sodium
citrate pH
6.0
15 min
0.35%
0.025%
T TBS
AUS 50
A.U.S.
3 min
0.35%
0.025%
T TBS
SERUM
BLOCK
3% BSA
in T
TBS
3% BSA
in T
TBS
3% BSA
in T
TBS
0.35%
0.025%
T TBS
3% BSA
in T
TBS
1:100
0.35%
0.025%
T TBS
10%
normal
goat
1:50
COUPE
N50
AUS
100
AUS
SG50
A.U.S.
A.U.S.
3 min
3 min
AUS
SG100
A.U.S.
3 min
0.35%
0.025%
T TBS
POS
BSA50
A.U.S.
3 min
0.35%
0.025%
T TBS
POS
SG50
A.U.S.
3 min
0.35%
0.025%
T TBS
H2O2
50
A.U.S.
3 min
standard
0.025%
T TBS
OI 50
A.U.S.
3 min
0.35%
0.025%
T TBS
OI 50
A.U.S.
3 min
0.35%
0.025%
T TBS
10%
normal
goat
3% BSA
in T
TBS
10%
normal
goat
3% BSA
in T
TBS
3% BSA
in T
TBS
3% BSA
in T
TBS
standard
0.025%
T TBS
3% BSA
in T
TBS
H2O2
50
A.U.S.
CB 50
Citrate
buffer pH
= 6,0
20 min
0.35%
0.025%
T TBS
LDH 50
A.U.S.
3 min
0.35%
0.025%
T TBS
LDH 1
A.U.S.
3 min
standard
0.025%
T TBS
3 min
29
3% BSA
in T
TBS
3% BSA
in T
TBS
3% BSA
in T
TBS
ANTIBODY
1:50
FINDINGS
Little background
staining No
membrane staining
Little background
staining Little
membrane staining
Little background
staining Little
membrane staining
Less background
staining and
membrane staining
than AUS 50
A lot of
background
staining especially
cytoplasmatic No
membrane staining
Less
backgroundstaining
than AUS SG50 no
specific membrane
staining
Merg; membrane
staining Cortex;
nucleusstaining
1:50
Negative
1:50
slide let loose
1:50
1:50
1:50
1:100
1:50
overnight
1:50
overnight
1:50
1:50
slide let loose
Little background
staining Little
membrane staining
Reasonable
background
staining Little
membrane staining
Little background
staining Little
membrane staining
1:50
Negative
1:50
Negative
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
ECH 1
A.U.S.
3 min
standard
0.025%
T TBS
AH/FH
1
A.U.S.
3 min
standard
0.025%
T TBS
PPVH 1
A.U.S.
3 min
standard
0.025%
T TBS
CH/CI 1
A.U.S.
3 min
standard
0.025%
T TBS
+/controle
A.U.S.
3 min
standard
0.025%
T TBS
30
3% BSA
in T
TBS
3% BSA
in T
TBS
3% BSA
in T
TBS
3% BSA
in T
TBS
3% BSA
in T
TBS
1:50
Little background
staining Little
membrane staining
Little background
staining Little
membrane staining
Little background
staining Little
membrane staining
Little background
staining Little
membrane staining
1:50
Negative
1:50
1:50
1:50
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Attachment C
stainingprotocol for Notch 1, paraffin embedded liver tissue, Rabbit anti-bodies:
Sc-6014 R
-Deparaffine serie:
-Xyleen 1, Xyleen 2
-Alc96%, Alc80%, Alc70%, Alc60%, Alc30%
-PBS
2 x 5 min.
5 x 5 min.
5 min.
-Rinse with Kimwipe cloth, circle slide with PAP pencil
-Various antigen retrievals
variable lengths
-H2O2 block;
5/15 min.
-Standard H2O2 DAKO or 0,35% H2O2
-Rinse in PBST
3 x 3 min.
-Treat with normal goat serum (1:10-1:30) in PBS in a humid chamber
30 min.
-Incubate with; -A; PBS (negative control)
-B; 6014-R rabbit (primary) antibody, dilution;
1:25- 1:500
(dilution range 1:50-1:500, Santa Cruz Biotechnology, Stored at 4 °C)
Overnight
------------------------------------------------------------------------------------------------------------Rinse in PBST
3 x 5 min.
-Incubate with Envision goat-anti-rabbit (secondary) antibody K4003
30 min. RT
-Rinse in PBS
3 x 5 min.
-Incubate with freshly made DAB, watch colour development; note time!
-- min.
-Rinse in running tap water
5 min.
-Counter stain with haematoxylin, 1:1 PBS
10 sec.
-Rinse in running tap water
5 min.
-Cover slide with Faramount aqueous mounting medium
For the different variables used in the protocol and their results see Attachment
A
31
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Attachment D
Stainingprotocol for Wnt (β-catenin), paraffin embedded liver tissue, AB 2982
-Deparaffine serie:
-Xyleen 1, Xyleen 2
-Alc96%, Alc80%, Alc70%, Alc60%, Alc30%
-PBS
2 x 5 min.
5 x 5 min.
5 min.
-Antigen retrieval
variable lengths
-Cooling down
variable lengths
-Rinse
2 x 5 min.
-Blocking;
30 min.
-Dry slides and take off as much liquid as you can
-Draw circles round coupe with ImmEdgde pen
-200 ul, or as much as you need to cover the coupes completely (humidity chamber),
with diluted 1st antibody;
-1:50 (20 μg/ml in 1% BSA, 0,025% Triton X-100 in TBS)
-Keep container at room temp
overnight
-------------------------------------------------------------------------------------------------------- ------------------- Rinse
2 x 5 min.
-Dry slides and take off as much liquid as you can
-H2O2 block;
-Standard H2O2 DAKO or 0,35% H2O2
5/15 min.
-2nd antibody at room temperature (Envision anti-Rabbit K4003)
30 min.
-Rinse
2 x 5 min.
-Dry slides and take off as much liquid as you can
-Incubate each slide with 200 µl DAB-solution
15 min. RT
-Put the sections in a container with milliQ water
-Dry slides and take off as much liquid as you can
-Cover sections for a few seconds with some drops of hematoxylin
-Flush sections with tap water
10 min.
Add enough drops aqueous mounting medium on each section and mount a cover glass
For the different variables used in the protocol and their results see Attachment
B
32
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Attachment E
Stainingprotocol for Envision method for anti CK7 Frozen sections
1. Place slide box in aluminium foil from -70 at RT
20 min.
2. Remove slides from slidebox and dry at air
10 min.
3. Rinse in PBS/Tween
3 min.
4. Block endogenous peroxidase activity with DAKO standard H2O2
30 min
5. Rinse in PBS/Tween
3x3 min
6. Treat with Normal Goat Serum 1:10 in PBS.
30 min
7. Incubate
a) with Mouse anti CK7 1:50
(K4001)
30 min RT
b) with PBS (negative control)
8. Rinse in PBS/Tween
3x5 min
9. Incubate with Envision Goat anti mouse
30 min RT
10. Rinse in PBS (without Tween)
3x5 min
11. Prepare DAB
12. Incubate in freshly made DAB
5 min.
13. Discard in jerrycan IV
Rinse 1x in tapwater in fumehood, discard in jerrycan IV
Rinse in running tapwater
5 min
16. Counter stain with haematoxylin
30 sec
20. Rinse in running tapwater
10 min
21. Dehydrate in 1x alc 60%- 1x alc 70% 2x alc 96%- 2x xylene
3 min. each step
22. Cover slides with Eukitt
33
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Attachment F
Protocol for Envision method on Frozen sections; Notch1
1. Place slide box in aluminium foil from -70 at RT
20 min.
2. Remove slides from slidebox and dry at air
10 min.
3. Rinse in TBS-0.025% Triton
3 min.
4. Block endogenous peroxidase activity with DAKO standard H2O2
5 min
5. Rinse in TBS-0.025% Triton
3x3 min
6. Treat with Normal Goat Serum 1:10 in TBS.
30 min
7. Incubate
A) Notch1; 6014-R rabbit 1:50 in TBS-0.025% Triton Overnight
B) rabbit serum 1:3000 in TBS-0.025% Triton
(negative control)
Overnight
8. Rinse in TBS-0.025% Triton
3x5 min
9. Incubate with Envision Goat anti rabbit K4003
45 min RT
10. Rinse in TBS (without 0.025% Triton)
3x5 min
11. Prepare DAB
12. Incubate in freshly made DAB
5 min.
13. Discard in jerrycan IV
Rinse 1x in tapwater in fumehood, discard in jerrycan IV
Rinse in running tapwater
5 min
16. Counter stain with haematoxylin 1:1 in TBS
10 sec
17. Rinse in running tapwater
10 min
18. Dehydrate in 1x alc 60%- 1x alc 70% 2x alc 96%- 2x xylene
3 min. each step
19. Cover slides with Vectamount
34
-MECHANISMS OF LIVER PROGENITOR CELL NICHE ACTIVATION IN CANINE LIVER DISEASE, DRS. D. LOOPSTRA-
Attachment G
Protocol for Envision method on Frozen sections; β-catenin
1. Place slide box in aluminium foil from -70 at RT
20 min.
2. Remove slides from slidebox and dry at air
10 min.
3. Rinse in TBS-0.025% Triton
3 min.
4. Block endogenous peroxidase activity with DAKO standard H2O2
5 min
5. Rinse in TBS-0.025% Triton
3x3 min
6. Treat with Normal Goat Serum 1:10 in TBS.
30 min
7. Incubate
Overnight
A) β-catenin; AB 2982 1:200 in TBS-0.025% Triton
B) rabbit serum 1:3000 in TBS-0.025% Triton
(negative control)
Overnight
8. Rinse in TBS-0.025% Triton
3x5 min
9. Incubate with Envision Goat anti rabbit K4003
30 min RT
10. Rinse in TBS (without Triton)
3x5 min
11. Prepare DAB
12. Incubate in freshly made DAB
5 min.
13. Discard in jerrycan IV
Rinse 1x in tapwater in fumehood, discard in jerrycan IV
Rinse in running tapwater
5 min
16. Counter stain with haematoxylin 1:1 in TBS
10 sec
17. Rinse in running tapwater
10 min
18. Dehydrate in 1x alc 60%- 1x alc 70% 2x alc 96%- 2x xylene
3 min. each step
19. Cover slides with Vectamount
35