Immunohistochemistry (IHC) Protocol-Paraffin Section Protocol


  • 1. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle.
  • 2. Rinse the tissue with running tap water for 30min-40min to eliminate the formaldehyde
  • 3. Dehydrate the tissues in EtOH baths in the following order: 70% Ethanol 20 min (x1); 95% Ethanol 20 min (x2); 100% Ethanol 20 min (x2)
  • 4. Clear the tissue in xylene for 2 times, 20 min each.
  • 5. Melt the paraffin prior to adding the tissue. Incubate the tissue in a 65 °C paraffin bath for 2 times, 30 min each.
  • 6. Pour melted paraffin into paraffin block mold. Place the tissue well in the mold and wait for its cooling down. (15-20 min)
  • 7. Section the paraffin-embedded tissue block in 4-10 μm thickness slides on a microtome and float in a 37°C water bath containing deionized water.
  • 8. Float the sections onto clean glass slides and microwave at 65°C for 15 min, then the tissue binds to the glass. Slides can be stored overnight at room temperature or be used in immunohistochemical staining immediately.


1. Paraffin section slides can be stored at room temperature for a long time.

2.Paraffin section is more suitble to reveal distribution of target antigen compared to frozen section.


Epitopes of target antigens are likely to be damaged by high temperature or fixative in the whole process and a antigen retrieval procedure is needed.