Thermo Fisher Scientific is committed to antibody performance and specificity testing. To support this commitment, each Invitrogen antibody that is indicated for immunohistochemistry applications has been tested using a protocol similar to that provided below. These tests help confirm antibody performance and help ensure superior results when used in experiments.

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 frozen sections that Invitrogen antibodies are subjected to is reproduced below. This protocol includes:

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.

Frozen tissue, indirect method

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. Air dry cut sections for 20 min.
  2. Fix sections by immersing in acetone for 10 min using a Coplin jar.
  3. Rehydrate the tissue in a Coplin jar with PBS or TBS for 10 min at room temperature.

    Note: From this point on, it is critical that the tissue does not dry out as this will result in high levels of background staining and difficulty interpreting staining results.

  4. Cover tissue with blocking reagent for 1 hr at room temperature (100 µL/ tissue section). Cover and place slide in a humidified, light-protected chamber.
  5. Uncover and immerse slide in a Coplin jar containing PBS or TBS. Using an orbital shaker set to low speed, gently agitate, changing PBS or TBS wash solution 2 more times for a total of 3 washes (5 min/wash).
  6. Dilute the primary antibody in blocking reagent according to the manufacturer’s recommended dilution. Overlay primary antibody solution on tissue and cover. Incubate in a humidified, light-protected chamber overnight at 4°C.
  7. Gently wash tissue 3 times in PBS or TBS (5 min/wash) as described in step 5.
  8. Dilute the secondary antibody in blocking reagent according to the manufacturer’s recommended dilution and protect from light.
  9. Incubate in a humidified, light-protected chamber for 1 hr at room temperature.
  10. Gently wash tissue 3 times in PBS or TBS (5 min/wash) as described in step 5.

    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.

  11. Mount and coverslip the slide. Seal the edge of the coverslip.
  12. Allow slides to dry for 1–2 hr before viewing on a microscope.
  13. Store at 4°C protected from light if needed.

Frozen tissue, direct method

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. Air dry cut sections for 20 minutes.
  2. Fix the sections by immersing in acetone for 10 minutes using a Coplin jar.
  3. Rehydrate the tissue in a Coplin jar with PBS or TBS for 10 min at room temperature.

    Note: From this point on, it is critical that the tissue does not dry out as this will result in high levels of background staining and difficulty interpreting staining results.

  4. Cover the tissue with blocking reagent for 1 hr at room temperature (100 µL/ tissue section). Cover and place slide in a humidified, light-protected chamber.
  5. Uncover and immerse the slide with the tissue into a Coplin jar containing PBS or TBS. Using an orbital shaker set to low speed, gently agitate, changing the PBS or TBS wash solution 2 more times for a total of 3 washes (5 min/wash).
  6. Dilute the antibody (or combination of multiple antibodies) in blocking reagent according to the manufacturer’s recommended dilution and protect from light. Cover and incubate in a humidified, light-protected chamber overnight at 4°C in the dark.
  7. Gently wash the tissue 3 times in PBS or TBS (5 min/wash) as described in step 5.

    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.

  8. Mount and coverslip the slide. Seal the edge of the coverslip.
  9. Allow slides to dry for 1–2 hr before visualizing.
  10. Slides can be stored at 4°C protected from light if needed.