IHC Core Guidelines and Instructions
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IHC Considerations:
An antibody that works for ELISA, Western or Flow Cytometry may not work for
IHC. If the antibody has not been tested with IHC before, it will take quite a bit of
effort to determine if the antibody can be used for IHC---optimizing will be
necessary for good results.
There should be a plan:
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Do you have tissue as negative and positive controls?
What will be the range of antibody dilutions and incubation times?
What will be the right blocking reagents?
What will be the secondary reagents?
Which antigen retrieval method? Heat induced antigen retrieval with
different pH or enzyme digestion?
Will a fluorescence OR enzyme detection system be used?
Will catalyzed signal amplification be needed?
Other Questions:
1. Are you going to immunostain frozen sections or paraffin sections?
2. What type of antibody is your primary antibody? A polyclonal rabbit?
Polyclonal goat? Mouse monoclonal or rat monoclonal?
3. What is the tissue you are testing: human or mouse tissue? From what organ?
4. Is the antibody commercially available and tested and shown to work well in
immunohistochemistry assays?
5. What is the expected Positive control tissue for the antibody?
6. What is the expected Negative control tissue for the antibody?
7. Do you have cells in tissue culture that you know are positive or negative for
the antibody that can be used as controls in the assay?
8. If the antibody has never been tested in IHC, what is the concentration of
antibody that you used on Westerns or ELISA to obtain the positive staining?
IHC Core Guidelines and Instructions
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IHC Core Guidelines
SLIDE POSITIONING:
Each slide contains 3 staining zones. The following total volumes of reagent are
recommended:
3 zones – 250 µL
2 zones – 200 µL
1 zone – 150 µL
1
2
3
Case #
Protocol
Zone #:
Note:
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The volumes may need to be increased for large human tissues.
For optimal results, the sections should be centered within each zone.
To save slides and reagents, we recommend placing experimental and
control tissues on the same slide for the same antibody. (The autostainer
cannot stain different antibodies on the same slide.) See examples of
tissue arrangement on the slides below:
Case #
Protocol
Block ID
Case #
Protocol
Block ID
KO#1
KO#2
WT#1
WT#2
positive ctrl
negative ctrl
experimental
PARAFFIN SECTIONS: 4-5 microns
Deparaffinization:
Slides should be dried overnight 37°C post-sectioning and prior to
deparaffinization, heated in oven at 60°C for 30-45 min in a horizontal position.
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Submerge slide in Slide Brite® (preferred safe non-hazardous method):
5min/change x 3 changes
OR
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Submerge slide in Xylene: 10 min/change x 2 changes
IHC Core Guidelines and Instructions
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Continue as below:
Rehydrate:
1. 100% ethanol 1 min
2. 95% ethanol 1 min – 2x
3. 70% ethanol 1 min
4. Leave in DI water until staining
Keep the slides in the DI water until ready to perform antigen retrieval. At no time
from this point onward should the slides be allowed to dry. Drying out will cause
non-specific antibody binding and therefore high background staining.
Deparaffinization should be done the night before or in the morning before you
drop off your slides.
FROZEN SECTIONS: 5 microns
Slides should be left to air dry overnight at room temperature immediately after
sectioning. If slides are not used the next day, wrap in foil and store in -20°C
freezer until use.
Fixation:
If slides are stored in the freezer, let the slides come to room temperature before
removing the foil.
1. Dip slides in Acetone: absolute alcohol (3:1) for 10-15 min at room
temperature
2. Immediately dip slides in TBS buffer
3. Keep slides in buffer until ready to stain
Slides from this point should not be allowed to dry.
This step should be done immediately before you send the slides to the core lab.
The following steps will be done in the IHC core lab:
A. Antigen Retrieval – Decloaking chamber (for paraffin sections)
Rodent decloaker – heat retrieval solution for rodent tissues
 ~ 95°C for 30-60 min
 ~ 120-125°C for 30 sec/5 min
Diva decloaker - heat retrieval solution for human tissues
 95-99°C for 30-40 min
Antigen retrieval for delicate tissues can use a lower temperature setting for a
longer period.
IHC Core Guidelines and Instructions
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B. Block endogenous peroxidase
 Peroxidazed 1
C. Protein block
 Human - Background Sniper
 Mouse – Rodent Block M
D. Immunostaining
Choosing the right reagent:
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Mouse-on-mouse HRP polymer kit
Rat-on-mouse HRP polymer kit
Rabbit-on-rodent HRP polymer kit
MicromerTM Universal HRP Detection kit
(for mouse anti-human or rabbit anti-human antibodies)
UltraVision LP Detection System – HRP polymer & DAB Plus Chromogen
The following chromogen kits are available:
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DAB (including if desired DAB Sparkle – DAB post-enhancing solution)
Romulin AEC
Fast Red
E. Counterstaining
 Hematoxylin (Blue, nuclei)
We will leave the slides in DI water for you to pick up. You can mount your slides
as follows:
G. Dehydration/Mounting
1. 70% ethanol 1 min.
2. 95% ethanol 1 min – 2x
3. 100% ethanol 1 min.
4. Xylene 10 min – 2x OR Slide Brite 2 min – 3x
5. Air dry
6. Coverslip