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Preparation and Fluorescent 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

Reagents

  • 4% paraformaldehyde
  • Xylene
  • 100% Ethanol, 95% Ethanol, 75% Ethanol, 50% Ethanol
  • 10 mM Sodium Citrate buffer (pH 6.0)
  • Permeabilization buffer
  • PBS
  • PBS-T: 0.4% Triton X-100 in PBS
  • Primary antibody
  • Secondary antibody

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. 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).
  2. Block any non-specific binding by incubating the tissue sections with 5% animal serum in PBS-T for 30 minutes at RT.

    3. 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 fluorescent label conjugated secondary antibody diluted with 1% serum in PBS-T. Incubate at RT for 1-2 hours.
  4. Wash sections twice with 1% serum PBS-T for 10 minutes each.

Step 5: Detection

  1. Tap off excess wash. Apply one drop of ant-fade mounting medium to the slide.
  2. Place a coverslip on the tissue sections. Circle the edges of the coverslip with clear fingernail polish to prevent the cells from drying. Allow the polish to air dry.
  3. Slides may now be examined under a microscope with the appropriate fluorescent filter sets. Be sure to limit slide exposure to light to prevent photobleaching.
  4. Slides can be stored between -20°C and 4°C in a dark slide box or slide book.

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