This immunohistochemistry protocol should be used as a guide for each researcher to build their own protocol, as each reagent will need to be optimized for use in the particular species, tissue type and application combination.
Ethanol, anhydrous denatured, histological grade (100% and 95%)
10X TBS: To prepare 1 liter of 10X TBS: 24.2g Tris base, 80g sodium chloride; adjust pH to 7.6 with HCl (use at 1X).
Wash Buffer TBS/T: 1X TBS, 0.1% Tween-20: To prepare 1 liter add 100ml of 10x TBS to 900 ml ddH2O. Add 1ml Tween 20 and mix.
10 mM Sodium Citrate Buffer: To prepare 1 liter, add 2.94g sodium citrate to 1 liter dH2O. Adjust pH to 6.0.
3% Hydrogen Peroxide: To prepare, add 10ml 30% H2O2 to 90ml dH2O.
Blocking Solution: 5% horse serum or goat serum in TBS
ABC Reagent: Prepare according to manufacturer’s instructions 30 minutes before use.
DAB Reagent: Prepare according to manufacturer’s recommendations.
A. Fresh Frozen Sections
Snap-freeze small tissue blocks (5 mm x 5 mm x 3 mm) in liquid nitrogen.
Transfer to cryostat, cut into thin (5–30 μm) sections, and transfer sections to positively charged slides (poly-L-lysine coated).
Dry the slides at RT (or 1-2 hours until completely dry if performing staining on the same day). Note: Thorough drying is required for proper adhesion to the slides.
A variety of fixation methods are available. Follow the specific methods mentioned in the product datasheet, or find the optimal method for your sample.
Cold acetone: 10 minutes at -20°C. Air dry.
Methanol: 10 minutes at -20°C.
10% Neutral buffered formalin: 10 minutes at RT.
3% Formaldehyde: 15 minutes at RT.
3% Formaldehyde/methanol: 15 minutes at RT, followed by 5 minutes in methanol at -20°C (do not rinse in between).
Wash slides 3 times, 5 min each in PBS, pH 7.4 containing 1% Tween 20.
B. Fixed, Frozen Tissue Sections
Perfuse tissue with fixative or immerse tissue in fixative for a set time period. 4% paraformaldehyde is the most commonly used fixative.
Immerse the tissue in cyroprotectant solution containing 10-30% sucrose in PBS. Cryoprotection is complete when the tissue no longer floats in the solution.
Remove tissue from the cyroprotectant solution and store at -70°C until sectioned.
Remove tissue from the -70°C freezer and equilibrate at -20°C for about 15 minutes before attempting to section. Equilibration helps prevent cracking of the block when sectioning.
Using a cryostat, prepare 10-15um sections that can be collected directly onto slides. Usually 3 sections can be placed per slide; spaced well apart.
Thoroughly dry sections on slides. Drying can be accomplished by air drying or by using a slide warmer, usually overnight, or at least 2-3 hours at 40-50°C.
Prepared slides can be stored dry at -70°C until stained. Equilibrate to room temperature and briefly re-dry prior to rehydration and staining.
C. Paraffin-Embedded Sections
Deparaffinize and hydrate sections
Xylene 2-3 changes, 5 minutes each.
100% absolute ethanol: 2 changes, 3 minutes each.
95% ethanol: 2 changes, 3 minutes each.
80% ethanol: 3 minutes.
50% ethanol: 3 minutes.
Distilled water, PBS, or Tris buffer: 2 changes, 3 minutes each.
Note: Once the tissue sections have been rehydrated, do not allow them to dry. Dry the slide around the tissue section with an absorbent wipe. Using a diamond pencil, pap pen, china marking pencil or fingernail polish, draw a circle on the microscope slide around the section. This circle will help retain solution on the section during subsequent incubations with reagents.
The visualization of many antigens can be significantly improved by pretreatment with antigen retrieval methods that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. Antigen retrieval techniques typically involve either the application of heat for varying lengths of time or the use enzymatic digestion by using proteases such as proteinase K, trypsin, or pepsin.
The heating methods typically utilize either a microwave oven, pressure cooker, steamer or water bath. Samples are heated for 20 minutes at close to 100°C, followed by cooling for an equivalent length of time. The most frequently used retrieval solutions are a) citrate buffer, pH 6.0, b) Tris-EDTA, pH 9.0 and c) EDTA, pH 8.0.
Perform antigen retrieval using Citrate Buffer if required:
Citrate buffer Recipe:
10mM sodium citrate, 0.05% Tween-20, pH 6.0 or
10mM citric acid, 0.05% Tween-20, pH 6.0
Pre-heat steamer or water bath with staining dish containing Sodium Citrate Buffer or Citrate Buffer until temperature reaches 95-100 °C.
Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user).
Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes.
Rinse sections in TBS-Tween-20 for 2×2 min.
Proceed to blocking step.
HYDROGEN PEROXIDE INCUBATION
Block endogenous peroxidase (if required):
Immerse slides into 0.3-3% H2O2 and 100% methanol for 10-30 minutes at RT.
Wash sections in distilled water, 2 changes for 5 minutes each.
Incubate sections with 3-10% normal serum from the same species as the secondary antibody, for 30 minutes to block non-specific binding of immunoglobulin.
Remove blocking solution.
Primary antibody incubation
*All steps should be performed in a moist environment.
Dilute the primary antibody in blocking solution. If no dilution is suggested, begin testing at 1:10, 1:100 and 1:1000.
Incubation overnight at 4°C.
Remove antibody solution. Wash 3 x 5 minutes in PBS, pH 7.4.
If the primary antibody is HRP-conjugated, proceed to Color Development.
Secondary antibody incubation
Dilute the biotin-conjugated secondary antibody in blocking solution according to the recommended dilution. Incubate 30-60 minutes at RT.
Removed secondary antibody solution. Wash slides 3 x 5 minutes in wash buffer.
Add streptavidin-horseradish peroxidase reagent to each section and incubate for 30 minutes at RT.
Remove ABC reagent. Wash sections 3x in wash buffer for 5 minutes each.
Apply the DAB solution to cover the sections completely in a moist environment. Incubate for 5-15 minutes at RT. Alternatively, observe the slide under a microscope to determine optimal color intensity of the insoluble precipitate.
As soon as color develops, stop the reaction by gently flushing with dH2O.
Counterstain tissue as desired with Hematoxylin & Eosin to define antigen proximity to normally expected structures.
Wash the slides with dH2O.
Dehydration and coverslip application
Dehydrate the sample for storage using a series of methanol or ethanol graded concentrations: 50% (2 x 5 min), 75% (2 x 5 min), and finally 100% (2 x 5 min).
Repeat in xylene, incubating sections two times for 10 seconds each.
Allow the slide to air dry.
Mount coverslips using appropriate mounting media.