Application Verification Testing for Immunohistochemistry (Paraffin)

Immunohistochemistry is a technique used to reveal the abundance, distribution, and localization of biomarkers within tissue. The biomarkers can be viewed directly using this technique, and the results offer insight into cell structure and mechanisms that are applicable for basic research.

The typical immunohistochemistry protocol for paraffin-embedded sections that Invitrogen antibodies are subjected to is reproduced below. This protocol includes:

• Formalin-fixed paraffin-embedded tissue, indirect method
• Formalin-fixed paraffin-embedded tissue, direct method

Note: Some of the steps in this protocol require optimization depending on the sample and antibody being used.

There are two detection methods for immunohistochemistry: colorimetric and fluorescent. Colorimetric detection involves the use of antibodies conjugated to an enzyme that produce a colored precipitate at the biomarker’s location. Fluorescent detection uses fluorescence-labeled antibodies, and results are visualized using fluorescence microscopy. Fluorescent detection also allows for the detection of multiple target antigens in the same tissue at the same time. Both detection methods can be used for direct or indirect immunohistochemistry.

Indirect is the most commonly used immunohistochemistry method. It provides greater signal amplification when the antigen is of low abundance, however there is potential for cross-reactivity from the secondary antibody. It is especially important when attempting indirect multiplexing to use primary antibodies raised in different species with different isotypes to reduce the change of cross-reactivity.

1. Cut paraffin-embedded tissue sections and mount the sections on slides. Place slides in vertical plastic slide holders.
2. Heat slides for 20 min at 50–60°C in a dry oven to facilitate attachment of tissue and soften the paraffin.
   
   

   Note: To prevent damage to target antigens temperature should not exceed 60°C.

3. Remove paraffin and rehydrate tissue using the following slide wash/incubation sequence:
   
   a) Histo-Clear II agent (3 x 5 min each)
   b) 100% ethanol (2 x 5 min each)
   c) 90% ethanol (5 min)
   d) 70% ethanol (5 min)
   e) ddH₂O (5 min)
   
   When moving the slides through the solutions, be sure to adequately mix the reagents and remove bubbles collecting on the slides by dipping the slide holder, with slides, up and down in the solution several times. From this point on, it is critical that the tissue does not dry out as this will lead to difficulty in interpreting staining results.
   
   
4. Completely submerge slides in excess amounts of a pre-heated antigen retrieval solution and microwave until boiling. Maintain a continuous boil for at least 15 min. After boiling, allow the slides to cool to room temperature (about 20 min) in the antigen retrieval solution.
5. Wash the slides in a Coplin jar with 1X PBS for 5 min using gentle agitation in an orbital shaker set to low speed.
6. Cover the tissue with blocking reagent for 1 hr at room temperature (100 μL/ tissue section). Cover and place slide in humidified, light-protected chamber.
7. Uncover and immerse the slide with tissue into a Coplin jar containing PBS. Using an orbital shaker set to low speed, gently agitate, changing the PBS wash solution 2 more times for a total of 3 washes (5 min/wash).
8. Dilute the primary antibody in blocking reagent according to the manufacturer’s recommended dilution. Overlay the primary antibody solution on the tissue and cover. Incubate in a humidified, light-protected chamber overnight at 4°C.
9. Gently wash the tissue 3 times in PBS (5 min/wash) as described in step 7.
   
   

   Note: If you are using an unconjugated or fluorescent primary antibody, continue to step 10 (3-step protocol). If you are using a biotinylated primary antibody, continue to step 12 (2-step protocol).

10. Dilute the secondary antibody in blocking reagent according to the manufacturer’s recommended dilution. If using a fluorophore-conjugated antibody, protect from light. Overlay the secondary antibody solution on the tissue and cover. Incubate in a humidified, light-protected chamber for 1 hr at room temperature.
11. Gently wash the tissue 3 times in PBS or TBS (5 min/wash) as described in step 7.
12. Dilute streptavidin conjugate in blocking reagent according to the manufacturer’s recommendation and protect from light. Overlay the streptavidin visualization reagent on the tissue and cover. Incubate in a humidified, light-protected chamber for 30 min at room temperature.
13. Gently wash the tissue 3 times in PBS or TBS (5 min/wash) as described in step 7.
    
    

    Optional: Nuclei can be counterstained using DAPI or DRAQ5. Ensure that you select a counterstaining agent that has a fluorescence emission spectrum that does not overlap with the emission spectrum of the other fluorophore(s) used in the experiment.

14. Mount and coverslip the slide. Seal the edge of the coverslip.
15. Allow slides to dry for 1–2 hr before viewing on a microscope.
16. Slides can be stored at 4°C protected from light if needed.

Direct detection is less common than indirect but has its own benefits. The benefits of direct detection include shorter sample staining times and the ability to use multiple antibodies raised in the same species.

1. Cut paraffin-embedded tissue sections and mount the sections on slides. Place slides in vertical plastic slide holders.
2. Heat slides for 20 minutes at 50–60°C in a dry oven to facilitate attachment of tissue and soften the paraffin.
   
   

   Note: To prevent damage to target antigens temperature should not exceed 60°C.

3. Remove paraffin and rehydrate tissue using the following slide wash/incubation sequence:
   
   a) Histo-Clear II agent (3 x 5 min each)
   b) 100% ethanol (2 x 5 min each)
   c) 90% ethanol (5 min)
   d) 70% ethanol (5 min)
   e) ddH₂O (5 min)
   
   When moving the slides through the solutions, be sure to adequately mix the reagents and remove bubbles collecting on the slides by dipping the slide holder, with slides, up and down in the solution several times. From this point on, it is critical that the tissue does not dry out as this will lead to difficulty in interpreting staining results.
   
   
4. Completely submerge slides in excess amounts of pre-heated antigen retrieval solution and microwave until boiling. Maintain a continuous boil for at least 15 min. After boiling, allow the slides to cool to room temperature (about 20 min) in the antigen retrieval solution.
5. Wash the slides in a Coplin jar with 1X PBS for 5 min using gentle agitation in an orbital shaker set to low speed.
6. Cover the tissue with blocking reagent for 1 hr at room temperature (100 μL/ tissue section). Cover and place slide in humidified, light-protected chamber.
7. Uncover and immerse the slide with tissue into a Coplin jar containing PBS. Using an orbital shaker set to low speed, gently agitate, changing the PBS wash solution 2 more times for a total of 3 washes (5 min/wash).
8. Dilute the fluorophore-conjugated primary antibody (or combination of multiple antibodies) in blocking reagent according to the manufacturer’s recommended dilution. Overlay the primary antibody solution on the tissue and cover. Incubate in a humidified, light-protected chamber overnight at 4°C.
9. Gently wash the tissue 3 times in PBS (5 min/wash) as described in step 7.
   
   

   Optional: Nuclei can be counterstained using DAPI or DRAQ5. Ensure that you select a counterstaining agent that has a fluorescence emission spectrum that does not overlap with the emission spectrum of the other fluorophore(s) used in the experiment.

10. Mount and coverslip the slide. Seal the edge of the coverslip.
11. Allow slides to dry for 1–2 hours before viewing on a microscope.
12. Slides can be stored at 4°C protected from light if needed.

Immunohistochemistry analysis of N-cadherin showing staining in the membrane of paraffin-embedded human heart tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a N-cadherin Mouse Monoclonal Antibody (Product # MA1-2002) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.