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.

Advantages:

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.

Disadvantages:

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.