The Immunohistochemistry Studies of
Tumor Necrosis Factor Αlpha and
Interferon Gamma Expression in
Pseudocapillaria tomentosa Infected and
Uninfected Zebrafish
Oregon State University
Lalee Lo
Dr. Jan Marie Spitsbergen
Dr. Susan Tornquist
What is immunohistochemistry?
Immuno- The immune system relies on
antibodies and antigen.
Histo- is the study of cells and tissue
Immunohistochemistry exploits the
characteristics of antibodies and antigens.
-Primary antibody attaches to Antigen
-Secondary antibody with tag attaches to
Primary Antibody.
What is immunohistochemistry?
Tumor Necrosis Factor Alpha
-apoptotic cell death
-cellular proliferation and differentiation
-inflammation
-regulation of immune cells
Interferon Gamma
-immune proteins produced in
response to challenges by:
-viruses
-bacteria
-parasites
-inflammation
Pseudocapillaria tomentosa
A common fish nematode known to cause:
-Inflammation of the gut and intestine of
zebrafish.
-Aggressive neoplasm (abnormal growth of
tissue) of the intestine.
Why Zebra Fish?
-Low maintenance and feed cost
-Ability to reproduce year round
-Zebra fish embryos develop rapidly,
progressing from eggs to larvae in under
three days and contain duplicate genes.
Current Research
-TNF α and IFN γ function in homeostasis.
-TNF α is produced by practically every cell.
-Higher levels of TNF α and IFN γ have
been observed in inflamed tissue.
Purpose of Research
To determine whether the levels of TNF α
and IFN γ elevate in response to parasitic
infection.
Goals and Objectives
Hypothesis:
Pseudocapillaria tomentosa infected
zebrafish would have stronger staining of
tissues with chromogen in
immunohischemistry studies indicating
elevated tissue levels of TNF α and IFN γ
Experiment Set #1
Date of
Birth
Date of
Carcinogen
Exposure
9/10/2006 None
9/10/2006 None
9/10/2006 None
9/10/2006 None
Carcinogen
Treatment
Date of
Parasite
Treatment
Parasite
Treatment
Lot #
Sampling
Date
# of Fish
None
5/10/200 Pseudoc
7
apillaria
ZRN
14-9
8/10/20
07
20
None
5/10/200 Pseudoc
7
apillaria
ZRN
14-10
8/10/20
07
20
None
5/10/200
7
None
ZRN
14-11
8/10/20
07
20
None
5/10/200
7
None
ZRN
14-12
8/10/20
07
20
Experiment Set #2
Date of
Birth
Date of
Carcinogen
Exposure
(age: 30
days)
Carcinogen
Treatment
Date of
Parasite
Treatment
Parasite
Treatment
Lot #
Sampling Date
# of
Fish
ZRN
14-5
5 fish at 3 wk and 5
wk, 20 fish at 17
wk, 30-50 fish at 29
wk post-parasite
74
1/5/2006 2/8/06
DMSO
Control
3/20/200 None
6
1/5/2006 2/8/06
DMSO
Control
3/20/200 Pseudocap ZRN
6 illaria
145b
See Above.
67
1/5/2006 2/8/06
DMBA 1
ppm
3/20/200 None
6
ZRN
14-6
See Above.
74
1/5/2006 2/8/06
DMBA 1
ppm
3/20/200 Pseudocap ZRN
6 illaria
146b
See Above.
90
Experiment Tank Feed Setup
Zebrafish were raised in flowing well water
in 30 gallon tanks at 27 Celsius +/- 2
-fed ad libitum with Aquatox (Zieger,
Gardners PA) flake fish feed and brine
shrimp.
-Necropies occurred at 11 months of age.
12 weeks post infection.
Methods of Analysis: Paraffin
Sectioning
Paraffin wax, is widely used on fruits,
vegetables, and candy to retard moisture
loss and spoilage.
The zebra fish tissue set in paraffin blocks,
this allows the tissue to be cut thin.
Methods of Analysis: Prep
Immuno-histochemistry Protocol (IHC):
-Xylene and ethanol. Dissolves the paraffin.
-Blocking solution
Binds to any sites that
are not antibody bound.
-Peroxidase Block
Block endogenous peroxidase.
Methods of Analysis: Protocol
Optimizing the IHC protocol:
-Finding the optimal TNF α and IFN γ
antibody concentration.
Antigen Retrieval Methods
-Enzyme vs Steam Antigen Retrieval
Steam Antigen Retrieval worked better.
Methods of Analysis: Protocol
Compared various buffers for the
steam heat treatment
-pH 6 citrate buffer
-Tris buffered saline (TBS) at pH 7.2
-Dako antigen retrieval buffer
For consistency reasons, we decided to
use the Dako antigen retrieval buffer.
Methods of Analysis: Antibody
ABCam TNF α stain
-Mouse derived antibody
ABCam IFN γ stain
-Rabbit derived antibody
DAKO Envision Plus Immunohistochemistry
polymer system
Methods of Analysis: Antibody
Methods of Analysis: Protocol
Primary Antibody
-Abcam TNF α (dilution 1:10,000)
-Abcam IFN γ (dilution 1:1,000)
Secondary Antibody
Chromogen (chemical compound that
changes color when bound)
Hematoxylin and then add mounting media.
Methods of Analysis: MPO
MPO (Myeloperoxidase) specifically stains
the cytoplasm of neutrophils of zebrafish.
Neutrophils produce reactive oxygen.
DNA damage likely plays a role in the
neoplasia associated with chronic
inflammation.
Results Myeloperoxidase
MPO expression found that the intestine of
zebrafish chronically infected with
Pseudocapillaria (3 months post infection
in experiment 2) showed increased
numbers of neutrophils compared to
intestine of uninfected fish.
Myeloperoxidase in the Intestine
MPO is negative in uninfected
intestine
MPO is positive in infected
intestine
Myeloperoxidase in Neutrophils in
Hematopoietic Tissue in the Kidney
MPO in neutrophils in impression
smear from kidney
MPO in neutrophils in
hematopoietic tissue of anterior
kidney
Results TNF α Experiment Set #1
No variation between infected and
uninfected group.
TNF α staining was strongest in:
-Head skeletal muscle
-Intestine
Experiment Set #1 TNF Alpha:
Tissue
Stained
Negative Control
Experiment Set #1 TNF Alpha: Eye
Stained
Negative Control
Experiment Set #1 Tumor
Necrosis Factor Alpha
Tissues Staining
Strongly
Tissues Staining Tissues Staining
Moderately
Lightly
Skeletal muscle of head
and at bases of fins
(derived from neural
crest)
Neurosensory epithelium
of nose
Bile duct mucosal surface
Intestine mucosal surface
(entire intestine)
Taste Buds
Exocrine pancreas acinar
cell
Ultimobranchial gland
Chloride cell of gill
filament and lamellae
Outer plexiform layer of
retina of eye
Heart ventricular
myocardium
Heart atrial myocardium
Results IFN γ Experiment Set #1
No variation between infected and
uninfected group.
IFN γ staining was strongest in:
-heart
-inflamed skeletal muscle and connective
tissue
Experiment Set #1 IFN Gamma:
Heart
Stained
Negative Control
Experiment Set #1 IFN Gamma:
Ventricle
Stained
Negative Control
Experiment Set #1 Interferon
Gamma
Tissues Staining
Strongly
Tissues Staining Tissues Staining
Moderately
Lightly
Endocardium of heart
Neurosensory epithelium of
nose
Pseudobranch epithelium
Macrophages in inflammation
in skeletal muscle
Taste Buds
Ameloblastic epithelium of
tooth
Inflammatory cells in
connective tissue
Hemopoietic tissue in
kidney (multifocal)
Meninges and ventricles of
brain
Chloride cells of gill
filament
Ganglia of cranial nerves
Developing oocytes
(perinucleolar and
vitellogenic)
Tooth pulp
Experiment Set #2 TNFα Elevation
Infected Intestine
Uninfected intestine. Red
chromogen indicates TNFα
expression.
Infected intestine. Red
chromogen indicates TNFα
expression.
Experiment Set #2 TNFα Elevation
Base of Dorsal Fin
Normal high
expression of TNFα
in skeletal muscle at
base of fin (short
arrow).
Lymphocytes and
macrophages in
inflamed skeletal
muscle (long
arrow).
High TNFα
expression in
capillaries in
inflamed skeletal
muscle
(arrowhead).
Experiment Set #2 IFNγ
Inflammation
M
M
M
M
M: Macrophages in inflamed
skeletal muscle
Arrow: Heart
Experiment Set #2 IFNγ
Inflammation
Arrow points to macrophages
expressing IFNγ in inflamed
skeletal muscle of trunk
Arrow points to macrophages
expressing IFNγ in skeletal
muscle near optic nerve (*)
Discussion
Experiment Set #1
Comparison of the infected vs uninfected
showed no difference in TNF α and IFN γ
at the 12 week post infection sampling.
Discussion
Experiment Set #2
5 weeks post infection showed there is a
difference in TNF α and IFN γ levels.
Experiment set #1 did not catch this
difference as the levels most likely
dropped after the peak earlier on.
Goals and Objectives
Hypothesis:
Pseudocapillaria tomentosa infected
zebrafish would have stronger staining of
tissues with chromogen in
immunohischemistry studies indicating
elevated tissue levels of TNF α and IFN γ
Conclusion
-TNF α and IFN γ is present in zebrafish
cells and function in cell homeostasis.
-No difference in TNF α and IFN γ levels
between infected and uninfected at the 12
weeks post infection.
-Higher levels appeared at 5 weeks post
infection.
Special Thanks to:
Mentor Dr. Jan Marie Spitsbergen
Additional Thanks
-Dr. Kate Fields
-Wanda Crannell
-Dr. Susan Tornquist
Additional Thanks
-The researchers at the Center for Fish
Disease Research.
-NIH (NIEHS)
-John Fryer Salmon Disease Lab
-Marine and Freshwater Biomedical Science
Center
-Environmental Health Science Center at
Oregon State University
Questions?