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Immunohistochemistry protocol

benchmark antibodies

Immunohistochemistry (IHC) protocol

  1. Tissue Preparation:
    • Collect the tissue samples of interest and fix them using an appropriate fixative (e.g., formalin or paraformaldehyde).
    • Trim the tissues into small sections and embed them in paraffin blocks or freeze them in optimal cutting temperature (OCT) compound.
  2. Tissue Sectioning:
    • If using paraffin-embedded tissues, cut thin sections (typically 4-6 μm) using a microtome and mount them on glass slides.
    • If using frozen tissues, cut thin sections (typically 10-20 μm) using a cryostat and mount them on glass slides.
  3. Deparaffinization and Rehydration:
    • If using paraffin-embedded sections, deparaffinize the slides by immersing them in xylene for 5-10 minutes, followed by a series of graded alcohols (100%, 95%, 70%) for rehydration. If using frozen sections, skip this step.
    • Wash the sections with running tap water for a few minutes to remove any residual chemicals.
  4. Antigen Retrieval:
    • Perform antigen retrieval if required, depending on the target antigen and fixation method. This step helps to expose the antigen epitopes for antibody binding.
    • Different antigen retrieval methods can be used, such as heat-induced epitope retrieval (HIER) using a microwave or pressure cooker, or enzyme digestion (e.g., proteinase K treatment).
    • Follow the specific instructions and optimal conditions for antigen retrieval based on the antigen and antibody manufacturer’s recommendations.
  5. Blocking:
    • Prepare a blocking solution such as 5% normal serum (from the same species as the secondary antibody) or 1% BSA in a suitable buffer (e.g., PBS or TBS).
    • Incubate the tissue sections with the blocking solution for 1-2 hours at room temperature to reduce non-specific binding.
  6. Primary Antibody Incubation:
    • Dilute the primary antibody specific to the target antigen in a suitable dilution buffer containing 1% BSA or serum.
    • Apply the primary antibody to the tissue sections and incubate overnight at 4°C or for 1-2 hours at room temperature in a humidified chamber.
  7. Washing:
    • Wash the tissue sections several times with a suitable washing buffer (e.g., PBS or TBS) to remove unbound primary antibody. Typically, 3-5 washes of 5 minutes each are sufficient.
  8. Secondary Antibody Incubation:
    • Dilute a species-specific secondary antibody conjugated to an enzyme (e.g., horseradish peroxidase) or a fluorescent dye in a suitable dilution buffer.
    • Apply the secondary antibody solution to the tissue sections and incubate for 1 hour at room temperature in a dark, humidified chamber.
  9. Washing:
    • Wash the tissue sections several times with a suitable washing buffer to remove unbound secondary antibody. Similar to the primary antibody washing step, 3-5 washes of 5 minutes each are generally recommended.
  10. Detection:
    • For enzyme-conjugated secondary antibodies, apply a chromogenic substrate (e.g., DAB or AEC) to visualize the target antigen. Incubate for a specific time, usually a few minutes, until the desired staining intensity is achieved.
    • For fluorescently-labeled secondary antibodies, mount the slides with an appropriate mounting medium containing a nuclear counterstain (e.g., DAPI) to visualize cell nuclei.
  11. Slide Coverslipping:
    • After detection, gently place a coverslip on top of the tissue sections using an appropriate mounting medium (e.g., aqueous or organic-based mounting media).
    • Ensure there are no air bubbles or debris between the coverslip and the tissue sections.
  12. Imaging and Analysis:
    • Examine the stained tissue sections under a microscope using the appropriate wavelength or filters for the detection method used (brightfield or fluorescence).
    • Capture images of the stained sections for documentation and analysis.
    • Analyze the staining pattern, intensity, and localization of the target antigen in the tissue sections.

Remember to always refer to the specific protocols and recommendations provided by the antibody and reagent manufacturers, as they may have specific instructions or variations for their products.