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IHC Protocol

15 September, 2023 by Anshul (neobio)

Hand in blue glove holding glass histology slides.

RE-Hydration Of Tissue Slides:

  • Label the tissue slides according to the protocol arrange them well in slide racks.
  • Bake slides in an oven at 60°C for 15 mins or until the paraffin on the tissue loosens and forms droplets.
  • Deparaffinize and rehydrate tissue sections by immersing slides through following series of solutions.
    Slide Brite 3 changes, 5 minutes each. (Slide Brite is a Xylene substitute)
    100% ethanol, 2 changes each
    95% ethanol, 2 changes 5 minutes each
    80% ethanol, 5 minutes
    50% ethanol, 5 minutes
    Distilled water, 2 changes 5 minutes each
    Hydrogen Peroxide 3%, 2 changes 5 minutes each.

Retrieval Of Antigen:

  • Antigen retrieval using BioCare’s Decloacker. Boil sections at 95°C for 45 minutes using Tris-EDTA (10mM Tris with 1mM EDTA, pH9.0) OR Citrate buffer (10mM Citrate pH6.0).


  • Use hydrophobic marker to create a barrier above and below tissue sections. Care must be taken in this step because sometimes marker can cause tissue dry if marking is near to the tissue. So, avoid close marking in this step.
  • Wash section twice, 3 times each in PBS.
  • Incubate primary antibody for 30 minutes.
  • Wash sections twice with PBS , 3-5 minutes each.
  • Incubate for 30 minutes with secondary antibody, goat anti-mouse / goat anti-rabbit-HRP polymer.
  • DAB 3 to 5 minutes Preparation of DAB: Take 950ul of DAB substrate add 500ul of DAB chromogen (ScyTek Laboratories). Mix well and use immediately.
  • Wash sections with PBS for 3-5 minutes, final wash should be DW.
  • Staining with Hematoxylin and bluing solutions Dip slide in hematoxylin 3-5 times and wash slides in tap water for 3 times. Dip slides in bluing solutions and wash slides in tap water for 3 times.
  • Observe cells under a microscope and find out the cellular localization in tissues.

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