Immunofluorescent IHC Staining of Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Protocol

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IHC Sample Preparation (Frozen vs. Paraffin-Embedded)

IHC Sample Fixation (Formalin vs. Alcohol)

IHC Blocking Non-Specific Binding

IHC Primary Antibody Selection

IHC Epitope Retrieval (HIER vs. PIER)

Secondary Antibodies Selection

HRP-Polymer Conjugates

IHC Detection Methods


IHC Paraffin Troubleshooting Guide

IHC Frozen Troubleshooting Guide

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Multicolor ICC/IF Staining

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PDF Download for Immunofluorescent IHC Staining of FFPE Tissue Protocol   Download Immunofluorescent Staining of Paraffin-Embedded Tissue protocol as a PDF



Tip: Before moving to alcohol grades step, make sure to completely deparaffinize the sections. If the sections still have traces of wax, an additional immersion of 5 minutes in Xylene may be employed.

  1. Deparaffinize and rehydrate by immersing the slides through the following solutions/wells:
    1. Xylene, three washes 5 minutes each
    2. 100% Ethanol, two washes 10 minutes each
    3. 95% Ethanol, two washes 10 minutes each
    4. 70% Ethanol, two washes 10 minutes each
    5. 50% Ethanol, two washes 10 minutes each
    6. Deionized Water, two washes for 5 minutes

  2. Draw a circle on the slide around the tissue with a hydrophobic barrier pen.


  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. Note: antigen retrieval conditions may require optimization. Read more about Antigen Retrieval for help determining optimal conditions for 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.


  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 room temperature.

Tip: The species of the animal serum used in permeabilization and blocking buffers is dependent on the host of your secondary antibody. (e.g. when using a goat anti-mouse secondary, use goat serum).


  1. Add primary antibody diluted in 1% animal serum PBS (with or without 0.05-0.1 % Triton X 100) to the sections and incubate at room temperature for 1-2 hours. Then store overnight at 4°C in humidified chamber. Use the recommended dilution of the antibody specified on the datasheet. If not specified, the typical starting dilution can be 2-5 µg/ml. For more information on primary antibody selection, please read our IHC Primary Antibody Selection Guide.

  2. Wash sections twice with 1% serum PBS-T for 10 minutes each.

  3. Add a fluorescent label conjugated secondary antibody diluted with 1% serum in PBS (with or without 0.05-0.1% Triton X 100) and incubate at room temperature for 1-2 hours. Use the recommended dilution of the antibody specified on the datasheet.
    Note: For help selecting the optimal secondary antibody, please read our Secondary Antibody Handbook.

  4. Wash sections twice with 1% serum PBS-T for 10 minutes each.


  1. Double Labeling: If using a second primary antibody and appropriately matched secondary, repeat step 5-8.

  2. Nuclear Labeling: After application of all primary antibodies, DNA binding dyes such as DAPI can be applied to the slides. After dye incubation, wash the slides once for 5 minutes with PBS.


  1. Tap off excess wash buffer and apply one drop of ant-fade mounting medium to the slide.

  2. Place a coverslip on the tissue sections and 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.