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Immunohistochemistry General Protocols



The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.








I. Background


II. Immunohistochemistry General Protocol

A. Materials Required
B. Fresh Frozen Sections
C. Fixed Frozen Sections
D. Paraffin-Embedded Sections


III. Antigen Retrieval

IV. Troubleshooting




I. Background

    Immunohistochemistry is widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. IHC staining is also widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme or cofactor, such as biotin, horseradish peroxidase (HRP) or alkaline phosphatase (AP) that can catalyze a color-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore. such as fluorescein (FITC) or R-Phycoerythrin (PE) (see Immunofluorescence).

    With the use of a conjugated primary antibody, direct IHC can be performed, but this method is seldom used because of the amplifying effect available with the use of a conjugated secondary antibody. The choice of secondary antibody for Immunohistochemistry is similar to that of ELISA and Western Blot. The conjugated secondary is directed specifically at the host species of the primary antibody and its specific isotype (e.g., mouse IgG1, goat IgM, rabbit IgG1,k, chicken IgY, etc.). In addition, if working with tissues or cells that have Fc receptors (thymus, spleen, blood, hematopoietic cells, leukocytes, B cells, etc.), it is best to choose an F(ab')2 fragment to eliminate non-specific binding through Fc receptors present on cells. As an alternative, binding of IgG whole molecule secondary antibodies to Fc receptors may be blocked by incubating cells in purified normal serum from the host species of the secondary antibody.


II. Immunohistochemistry General Protocol

A. Materials required:

1. Acetone

2. Ethanol, anhydrous denatured, histological grade (100% and 95%)

3. dH2O

4. Hematotoxylin

5. Xylene

6. 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).

7. Wash Buffer TBS/T: 1X TBS, 0.1% Tween-20: To prepare 1 liter add 100ml of 10x TBS to 900ml ddH2O. Add 1ml Tween 20 and mix.

8. 10mM Sodium Citrate Buffer: To prepare 1 liter, add 2.94g sodium citrate to 1 liter dH2O. Adjust pH to 6.0.

9. 3% Hydrogen Peroxide: To prepare, add 10ml 30% H2O2 to 90ml dH2O.

10. Blocking Solution: 5% horse serum or goat serum in TBS

11. ABC Reagent: Prepare according to manufacturer's instructions 30 minutes before use.

12. DAB Reagent: Prepare according to manufacturer's recommendations.


B. Immunohistochemistry (Fresh Frozen Sections)

1. Tissue preparation:

   a. Snap-freeze small tissue blocks (5x5x3mm) in liquid nitrogen.

   b. Transfer to cryostat, cut into thin (5–30um) sections, and transfer sections to positively charged slides (poly-L-lysine coated).

   c. 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.


2. Fixation methods

   a. 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).


   b. Wash slides 3 times, 5 min each in PBS, pH 7.4 containing 1% Tween 20


C. Fixed, Frozen Tissue Sections

1. Perfuse tissue with fixative or immerse tissue in fixative for a set time period. 4% paraformaldehyde is the most commonly used fixative.

2. Immerse the tissue in cyroprotectant solution containing 10-30% sucrose in PBS. Cryoprotection is complete when the tissue no longer floats in the solution.

3. Remove tissue from the cyroprotectant solution and store at -70°C until sectioned.

4. 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.

5. 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.

6. 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.

7. Prepared slides can be stored dry at -70°C until stained. Equilibrate to room temperature and briefly re-dry prior to rehydration and staining.


D. Paraffin-Embedded Sections

1. Deparaffinize and hydrate sections

    a. Xylene 2-3 changes, 5 minutes each

    b. 100% absolute ethanol: 2 changes, 3 minutes each

    c. 95% ethanol: 2 changes, 3 minutes each

    d. 80% ethanol: 3 minutes

    e. 50% ethanol: 3 minutes

    f. 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, 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.


2. Antigen Retrieval

    a. Perform antigen retrieval if required. (For antigen retrieval methods see Section V. HIER (heat-induced epitope retrieval) using citrate buffer is a commonly used method.

3. Hydrogen Peroxide incubation:

    a. Block endogenous peroxidase (if required) by immersing slides into 0.3-3% H2O2 and 100% methanol for 10-30 minutes at RT.

    b. Wash sections in distilled water, 2 changes for 5 minutes each.


E. Immunostaining

1. Blocking step:

    a. 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.

    b. Remove blocking solution.

2. Primary antibody incubation
(All steps should be performed in a moist environment.)

    a. Dilute the primary antibody in blocking solution. If no dilution is suggested, begin testing at 1:10, 1:100 and 1:1000.

    b. Incubation overnight at 4°C.

    c. Remove antibody solution. Wash 3 x 5 minutes in PBS, pH 7.4.

    d. If the primary antibody is HRP-conjugated, proceed to Color Development.

3. Secondary antibody incubation

    a. Dilute the biotin-conjugated secondary antibody in blocking solution according to the recommended dilution. Incubate 30-60 minutes at RT.

    b. Removed secondary antibody solution. Wash slides 3 x 5 minutes in wash buffer.

4. Color Development

    a. Add streptavidin-horseradish peroxidase reagent to each section and incubate for 30 min. at RT.

    b. Remove ABC reagent. Wash sections 3x in wash buffer for 5 min. each.

    c. 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.

    d. As soon as color develops, stop the reaction by gently flushing with dH2O.

    e. Counterstain tissue as desired with Hematoxylin & Eosin to define antigen proximity to normally expected structures.

    f. Wash the slides with dH2O.

5. Dehydration and coverslip application

    a. Dehydrate the sample for storage using a series of methanol or ethanol graded concentrations: 50% (2x5min), 75% (2x5min), and finally 100% (2x5min).

    b. Repeat in xylene, incubating sections two times for 10 seconds each.

    c. Allow the slide to air dry.

    d. Mount coverslips


III. Antigen Retrieval

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.

Antigen Retrieval using Citrate Buffer:
Citrate buffer:
10mM sodium citrate, 0.05% Tween-20, pH 6.0 or
10mM citric acid, 0.05% Tween-20, pH 6.0

1. Pre-heat steamer or water bath with staining dish containing Sodium Citrate Buffer or Citrate Buffer until temperature reaches 95-100 °C.

2. 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).

3. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes.

4. Rinse sections in TBS-Tween-20 for 2x2 min.

5. Proceed to blocking step.


IV. Troubleshooting

A. Problem: Weak or no staining:

1. Antibodies do not work due to improper storage: Aliquot antibodies into smaller volumes and store in freezer (-20°C) and avoid repeated freeze/thaw cycles; store antibodies according to manufacturer's instructions.

2. Antibody concentration was too low: Increase the concentration of primary and/or secondary antibodies. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noise ratio.

3. Inadequate antibody incubation time: Increase antibody incubation time.

4. Inadequate or improper tissue fixation: Increase duration of postfixation or try different fixatives.

5. Incompatible secondary and primary antibodies: Use secondary antibody that will interact with primary antibody. For example, if primary antibodies are raised from rabbits, use anti-rabbit secondary antibodies.

6. Inactive secondary antibody: Replace with a new batch of antibody.

7. Inactive streptavidin-horseradish peroxidase reagent: Replace with a new batch of reagents.

8. Defective or incompatible enzyme substrate (DAB) system: Replace with a new batch of reagents.

9. Inadequate substrate incubation time: Increase the substrate incubation time.


B. Problem: High Background

1. Inadequate washing of sections. Wash at least 3x between steps.

2. Tissue contains endogenous enzyme such as peroxidase or alkaline phosphatase. Block endogenous enzyme activities using 3% hydrogen peroxide (blocks peroxidase) in methanol or levamisole (blocks AP) prior to incubation with primary antibody

3. Tissue contains endogenous biotin activity. Block endogenous biotin activity using the avidin-biotin blocking reagent prior to incubation with primary antibody.

4. Mouse antibody used on mouse tissues. Treat tissue with mouse on mouse blocking reagent prior to the primary antibody incubation.

5. Sections dried out. Avoid letting sections dry out.