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Preparation and Chromogenic IHC Staining of Paraffin-embedded Tissue Sections

Contents

Step 1: Deparaffinization and Rehydration

Step 2: Antigen Retrieval

Step 3: Permeabilization and Blocking Non-Specific Binding

Step 4: Antibody Staining

Step 5: Detection

Step 6: Dehydration and Mounting

Reagents

Methods

Step 1: Deparaffinization and Rehydration

  1. Deparaffinize and rehydrate by immersing the slides through the following solutions/ wells:
    • Xylene, two times for 10 minutes each
    • 100% Ethanol, two times for 10 minutes each
    • 95% Ethanol, two times for 10 minutes each
    • 70% Ethanol, two times for 10 minutes each
    • 50% Ethanol, two times for 10 minutes each
    • Deionized water, two times for 5 minutes each
  2. Draw a circle on the slide around the tissue with a hydrophobic barrier pen.

Step 2: Antigen Retrieval

  1. For heat induced antigen retrieval, using a microwave, bring the slides to a boil in 10 mM Sodium Citrate buffer (pH 6.0) and then maintain at a sub-boiling temperature for 10 minutes.

    2. Note: antigen retrieval conditions may require optimization depending on your sample.

  2. Let the slides cool on the bench-top for 30 minutes.
  3. Wash the sections by immersing them in distilled water for 5 minutes each.

Step 3: Permeabilization and Blocking Non-Specific Binding

  1. For endogenous peroxidase activity block, quench the tissue sections with 3.0% hydrogen peroxide in methanol for 15 minutes.
  2. Wash the sections in distilled water two times for 5 minutes each.
  3. To permeabilize the tissue/cells, wash the sections twice for 10 minutes each with permeabilization buffer containing 1% animal serum and 0.4% Triton X-100 in PBS (PBS-T).
  4. Block any non-specific binding by incubating the tissue sections with 5% animal serum in PBS-T for 30 minutes at RT.

    5. Note: The species of the animal serum used is dependent on the host of your secondary antibody. For instance, when using a goat anti-mouse secondary, use goat serum.

Step 4: Antibody Staining

  1. Add the primary antibody diluted in 1% animal serum in PBS-T to the sections. Incubate at RT for 1-2 hours. Continue the incubation overnight at 4°C.

    2. Note: The recommended starting dilution is 2-5 µg/ml if there are not specified antibody dilution.

  2. Wash sections twice with 1% serum in PBS-T for 10 minutes each.
  3. Add a HRP conjugated (HRP-DAB detection method) or biotinylated secondary antibody (ABC-HRP-DAB detection method) to each section. Incubate the sections at RT for 1 hour.
  4. Wash sections twice with 1% serum PBS-T for 10 minutes each.

Step 5: Detection

  1. For the ABC method, add ABC-HRP reagent to each section. Incubate at RT for 1 hour. For HRP-DAB method, skip ABC-HRP step and move to DAB incubation step.
  2. Prepare a working solution of DAB and apply to tissue sections.

    3. Note: Monitor the reaction as the chromogenic reaction turns the epitope sites brown. Time of color development may vary from few seconds to 10 minutes.

  3. As soon as the section develop, immerse them in deionized water for 2 minutes each.
  4. If nuclear counterstaining is desired, use Hematoxylin according to the manufacturer’s instructions.

If you are using an aqueous chromogen instead of DAB such as Fast Red, AEC, skip the following Step 6 and mount in aqueous media instead of organic mounting media.

Step 6: Dehydration and Mounting

  1. Dehydrate tissue sections by moving slides through the following solutions twice for 2 minutes each: 95% Ethanol, 100% Ethanol, Xylene.
  2. Add mounting media to slides and top with coverslips. The DAB reaction is permanent and stable and can be analyzed under a brightfield microscope at any time.
Related Sections

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