Preparation Of Paraffin-Embedded Sections For Immunohistochemistry

Sectioning

Tissue sections (5-7 µm thick) are cut from paraffin-embedded blocks on a microtome and mounted from warm water (40°C) onto adhesive microscope slides. Sections are allowed to dry overnight at room temperature or 40°C. CAUTION: ANTIGENS MAY BE DESTROYED BY EXPOSURE TO HIGH TEMPERATURES. (Adhesion of the section to the slide is essential to prevent tissue loss during subsequent incubations and washes).

Deparaffinization

Deparaffinize and rehydrate tissue sections using standard methods. It is necessary to completely remove the embedding material. The xylene and alcohol bath solutions should be replaced frequently.Suggested Reagents and Times for Deparaffinization
Xylene 100% alcohol

95% alcohol

80% alcohol

70% alcohol

Phosphate buffered saline or Tris buffer 2 to 3 changes, 5 minutes each 2 changes, 3 minutes each

2 changes, 3 minutes each

3 minutes

3 minutes

2 changes, 3 minutes each

PRECAUTION: 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.

Immunostaining StepsTap off excess buffer and wipe around the tissue section with an absorbent wipe. CAUTION: DO NOT TOUCH THE TISSUE OR ALLOW IT TO DRY.

Antigen retrieval (optional, see Antigen retrieval in Appendix). 

Immerse slides into 0.3-3% H2O2 and 100% methanol for 10 minute at RT to quench endogenous peroxidase.
Wash slides in cold buffer for 2 changes of 5 to 10 minutes each.
Apply one or two drops of pre-blocking agent to the tissue section and allow it to remain in place for 10 to 20 minutes or according to the manufacturer’s instructions (see Pre-blocking Agents in Appendix).
Tap off the pre-blocking agent and wipe away any excess around the tissue. CAUTION: DO NOT TOUCH THE TISSUE OR ALLOW IT TO DRY.
Block biotin/avidin (optional, see Pre-blocking Agents in Appendix). 
Tap off the blocking agents and wipe away any excess around the tissue. 
Cover each section with one or two drops of diluted primary antibody or negative control incubate the slides at room temperature for 1 hour or 4°C for 12 to 24 hours in a closed incubation chamber. OPTIMAL conditions must be determined empirically.
Remove the slides from the incubation chamber and rinse the excess antiserum from the slide with a gentle stream of cold PBS or Tris buffer from a wash bottle. Continue washing sections by immersing in cold buffer for 2 changes of 5 to 10 minutes each
Blot the excess buffer from the slide with an absorbent wipe. CAUTION: DO NOT TOUCH THE TISSUE OR ALLOW IT TO DRY. Cover each section with one to two drops of diluted secondary antibody. Incubate the slides in an incubation chamber at room temperature for 1 hour. OPTIMAL conditions must be determined empirically.
Remove the slides from the incubation chamber and rinse the excess antiserum from the tissue with a gentle stream of room temperature PBS or Tris buffer. Continue washing sections by immersing in room temperature wash buffer for 2 changes of 5 to 10 minutes each.
If using biotinylated secondary antibodies, repeat step 12 using SA-HRP reagent diluted PBS. Incubate 30 minutes at room temperature.
Immerse the tissue sections in a freshly prepared chromogen/substrate reagent solution such as DAB/H2O2 or AEC/H2O2 for 2 to 7 minutes. Remove slides periodically to check for staining development with a low power microscope. Stop stain by rinsing with PBS or TBS.
If organic-based mounting media is to be used, dehydrate sections with increasing concentrations of ethanol followed by xylene as in conventional histology. CAUTION: AEC IS ORGANICALLY SOLUBLE, DO NOT DEHYDRATE SECTIONS.

APPENDIX

Equipment For Sectioning Microtome

Supplies histological staining dishes wash bottles absorbent wipes coverslips

Reagent Materials Required primary antibody for immunohistochemistry secondary antibody: an antibody, conjugated with biotin or HRP, against a species immunoglobulin that matches the host species of the primary antibody. Streptavidin-HRP: This enzyme conjugate is needed if a biotinylated secondary antibody is used. Negative or isotype Control: Normal serum from the same animal or species providing the primary antiserum or the same immunoglobulin isotype. Prepare same as primary antiserum; use same diluent and working dilution. Antisera Diluent: Use to dilute primary and secondary antisera
Phosphate buffered saline (PBS), pH 7.4 or Tris/Saline buffer (TBS), pH 7.6. In some cases, 0.25% bovine serum albumin may be added to reduce background. Antigen retrieval Agents: Certain paraffin-embedded sections need to have antigen retrieval procedure to uncover antigenic sites from tissue sections. Commonly used methods are either microwave heat treatment by boiling the sections in 0.01M citrate buffer (pH 6.0) for 10 –20 minutes or enzyme digestion by incubating sections with a proteolytic enzyme (such as trypsin (0.05% (v/v) in PBS with 0.1% CaCl2) at 37°C, or at room temperature for 10 – 20 minutes. The end users must control the conditions of concentration, time and temperature. Pre-blocking Agents: A reagent solution that serves to block nonspecific antigenic sites found on tissue. H2O2 methanol blocking solution: Contains 0.3-3% H2O2 and 100%methanol to block endogenous peroxidase.

Normal serum blocking solution: Contains normal serum from host species providing second antibody. 1-10% normal serum in combination with 0.25% bovine serum albumin in PBS pH 7.4 or Tris buffer pH 7.6. Block may contain detergent if antigens are internal (0.1-0.3% triton X-100 are typical).

Biotin/Avidin blocking solution: It is necessary to use a commercially available kit to block endogenous biotin and avidin when using a biotinylated secondary antibody on certain tissues that are rich in biotin and avidin such as liver, kidney and small intestine. Rinsing/Washing Buffer PBS, pH 7.4 Tris buffer pH 7.6 Chromogen DAB dissolved in Tris buffer pH 7.6 AEC dissolved in acetate buffer. CAUTION: the chromogens are carcinogenic; please handle and dispose of in an appropriate manner in compliance with local and federal regulations Substrate reagent Hydrogen peroxide Dehydration reagents (for DAB). Ethanol baths of increasing concentration: 70, 80, 95, and 100% ethanol and xylene baths Mounting Media Water-based mounting media (can be used with both DAB and AEC). Permanent organic-based mounting media (for DAB only). Tissue must be dehydrated prior to use.