Locating Gene Mutations
Associated With Breast Cancer Using
IHC (ImmunoHistoChemistry)
http://www.abcam.co.jp/nqo1-antibody-ab34173.html
Purpose
• IHC can be used to detect mutations in genes
that are associated with the development of
breast cancer, and the risk of developing
breast cancer.
• IHC can also detect HER2 hormone receptors
on the tumors surface, which rates it on a
scale form 0 to 3+.
Purpose
• IHC tests can tell a persons risk for developing
breast cancer, pinpoint the specific mutated
gene, and detect the levels of hormone
receptors.
• All of these results greatly impact the patients
treatment and preventative care.
Gene Mutations Related to Increased
Risk of Breast Cancer
•
•
•
•
•
•
BRCA1
BRCA2
CDH1
PTEN
STK11
TP53
Gene Mutations Associated With
Breast Cancer
•
•
•
•
•
•
•
•
•
•
•
AR
ATM
BARD1
BRIP1
CHEK2
DIRAS3
ERBB2
NBN
PALB2
RAD51
RAD50
BRCA1 in Human Breast Cancer
www.rndsystems.com/images/ihc/af2210_2.jpg
BRCA1
• Detection of Breast Cancer gene 1 (BRCA1) in paraffinembedded human breast cancer tissue sections using 5
µg/mL of R&D Systems goat anti-human affinitypurified BRCA1 polyclonal antibody. Tissues were
stained using anti-goat HRP-DAB detection reagents
(brown) and counterstained with hematoxylin (blue).
• This antibody specifically recognizes human BRCA1.
This antibody has not yet been tested in other species.
This antibody can be used for immunohistochemistry,
immunoprecipitation, Western blot, and direct ELISA.
Troubleshooting
Possible Source
Lack of antigen
Test or Action
Check protein expression by in situ hybridization (in
some rare cases translation may be blocked even
though mRNA is detected)
Antibodies do not work due to improper
storage
Aliquot antibodies into smaller volumes sufficient to
make a working solution for a single experiment. Store
aliquots in a freezer (-20 to -70°C) and avoid repeated
freeze-thaw cycles
Inactive primary antibodies
Replace with a new batch of antibodies
Inadequate tissue fixation
Try different fixative
Incompatible secondary and primary
antibodies
Use secondary antibody that will interact with the
primary antibody. For example, if primary antibodies
were raised in rabbits, use anti-rabbit secondary but not
anti-mouse or anti-goat
Inactive secondary reagents
Use secondary antibody that will interact with the
primary antibody. For example, if primary antibodies
were raised in rabbits, use anti-rabbit secondary but not
anti-mouse or anti-goat
Inactive secondary reagents
Replace with new reagents
Antigen was destroyed before incubation
with primary antibody
Quenching of endogenous peroxidase was done prior to
the addition of primary antibodies. Block peroxidase
after incubation with primary antibody
Overstating
Possible Source
Test or Action
High concentration of
primary and/or secondary
antibodies and/or reagent
Determine optimal concentration for
each component of
immunohistochemical reaction: primary
antibodies; secondary antibodies;
enzymes catalyzing formation of color
precipitates
Long incubation time
Determine optimal incubation time for
each component of
immunohistochemical reaction: primary
antibodies; secondary antibodies;
enzymes; chromogenic substrates
Non-specific binding of
primary and/or secondary
reagents to tissues
Treat tissues to reduce or block nonspecific binding of immunohistochemical
components to tissues, for example, by
Avidin-Biotin Blocking Reagents
High Background
Possible Source
Test or Action
Incubate with normal serum obtained from
species other than from the species that
were the source of the primary antibody
Tissues have endogenous
molecules which are also used in
incubation mixtures, for example,
the presence of peroxidase in blood
cells or the presence of
autofluorescent pigment lipofuscin
in neuronal cells
Block endogenous component. Endogenous
peroxidase may be blocked by incubating
tissues for 30 minutes at room temperature
with 0.3% H2O2 in methanol, or switch to
protocols utilizing substances which are not
present in tissues
For autofluorescence: after finishing IHC
staining, treat tissues with 1% Sudan Black
in 70% alcohol. If residual autofluorescence
still obscures specific labeling, use nonfluorescent enzymatic protocols with
chromogenic substrates (for instance DAB
or AEC) or employ immunogold-silver
staining
Procedure
Sample Preparation (Frozen Tissues)
The vast majority of immunohistochemical procedures employ a cell or tissue
fixation step using formaldehyde or other cross-linking fixatives prior to
incubation with primary antibody. Fixation is required to retain tissue
morphology and prevent degradation of tissue antigens. Fixation may be
performed either by immersing dissected pieces of tissue (e.g. human
biopsies) into the fixative, or by vascular perfusion (e.g. laboratory animals
such as mice, rats, guinea pigs, etc.). It is very important to optimize fixing
conditions since under- or over-fixation may reduce or abolish tissue
immunoreactivity. The easiest way to correct under-fixation is to post-fix
tissue sections on the slide before starting immunohistochemical staining.
To recover antigens in over-fixed tissues, either protease-induced epitope
retrieval (PIER) or heat-induced epitope retrieval (HIER) techniques are
recommended. HIER can be performed using a microwave oven, pressure
cooker, vegetable steamer, autoclave or water bath. After tissues are fixed,
they may either be embedded into paraffin or covered with OCT
compound and frozen for further sectioning. Paraffin-embedded tissues
are cut using a microtome at room temperature, whereas frozen tissues
are cut using a cryostat at temperatures below 0° C. Antigen
immunoreactivity was found to be better preserved in frozen rather than
paraffin-embedded tissues.
Procedure
Tissue Fixation and Mounting:
1. Fix the tissue by vascular perfusion with 500 - 700 mL of Fixative (-10% Neutral
Buffered Formalin)
2. Dissect the tissue.
3. Immerse the tissue in 70% ethanol three times for 30 minutes each at room
temperature.
4. Immerse the tissue in 90% ethanol two times for 30 minutes each at room
temperature.
5. Immerse the tissue in 100% ethanol three times for 30 minutes each at room
temperature.
6. Immerse the tissue in toluene three times for 20 minutes each at room
temperature.
7. Embed the tissue in paraffin (Paraplast, Fisher Scientific) two times for 60
minutes each at 58° C. Alternatively, tissues can be embedded into paraffin using
specialized automated tissue processing systems.
8. Cut 5 - 15 µm thick tissue sections using a rotary microtome.
9. Float the sections in a 56° C water bath.
10. Mount the sections onto histological slides.
11. Dry the slides overnight at room temperature. Slides containing paraffinembedded sections can be stored at room temperature.
Procedure
• When it is not possible to fix tissue by perfusion,
dissected tissue may be fixed by immersing the
tissue into a 10% formalin solution for 4 - 8 hours
at room temperature. It is commonly accepted
that the volume of fixative should be 50 times
greater than the size of the immersed tissue.
Avoid fixing the tissue for greater than 24 hours
since tissue antigens may either be destroyed or
masked (A.C. Cuello, ed., 1993,
Immunohistochemistry: Methods in the
Neurosciences, Vol. 14; IBRO Handbook Series,
John Wiley & Sons, New York).
Procedure
Antigen Retrieval Before Staining:
Most formalin-fixed tissue requires an antigen retrieval step
before immunohistochemical staining can proceed.
This is due to the formation of methylene bridges during
fixation, which cross-link proteins and therefore mask
antigenic sites. The two methods of antigen retrieval are heatmediated (also know as heat-induced epitope
retrieval, or HIER) and enzymatic.
Antigen retrieval with Tris/EDTA pH 9.0 buffer is suitable for
most antigens. Sodium citrate pH
6.0 is also widely used.
Procedure
1. Insert the slides containing tissues into the heated
Retrieval Solution and incubate 2 - 10 minutes. Note:
cryostat sections are more susceptible to the damaging
effects of the Retrieval Solution than paraffin-embedded
tissues.
2. Place the Coplin jar containing the Retrieval Solution and
slides on a lab bench and allow to cool for 5 - 10 minutes
at room temperature.
3. Rinse the slides with distilled water followed by PBS. Note:
tissues may become loose after the retrieval procedure,
avoid vigorous rinsing to prevent detachment of the
tissues from the slides.
Procedure
Staining:
Day 1
1. Wash the slides 2 x 5 minutes in TBS plus 0.025% Triton X100 with gentle agitation.
2. Block in 10% normal serum with 1% BSA in TBS for 2
hours at room temperature
3. Drain slides for a few seconds (do not rinse) and wipe
around the sections with tissue paper
4. Apply primary antibody diluted in TBS with 1% BSA
5. Incubate overnight at 4°C
Procedure
Day 2
1. Rinse 2 x 5min TBS 0.025% Triton with gentle agitation.
2. If using an HRP conjugate for detection, incubate the slides in 0.3% H2O2 in TBS for
15 min
3. For enzymatic detection (HRP-DAB):
Apply enzyme-conjugated secondary antibody to the slide diluted to the concentration
recommended by
the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature.
4. Rinse 3 x 5min TBS.
5. Develop with chromogen for 10 min at room temperature
6. Rinse in running tap water for 5 min.
7. Counterstain, if desired, with hematoxylin for 1-5 seconds. Slides are then air dried
and can be mounted and coverslipped with Immunohistochemistry Mounting
Medium without any further dehydration. Slides that need to be kept for long
periods in optimal shape can be dehydrated in ethanol and mounted in special
mounting medium.
8. Dehydrate, clear and mount
References
• http://www.jhc.org/cgi/rapidpdf/jhc.7A7209.200
7v1.pdf
• http://ghr.nlm.nih.gov/condition=breastcancer
• http://www.breastcancer.org/symptoms/testing/
types/ihc.jsp
• http://www.abcam.com/ps/pdf/protocols/ihc_p.
pdf
• http://www.rndsystems.com/ihc_detail_objectna
me_EnzymaticProtocol20.aspx