Formaldehyde fixation impairs or totally destroys the immunoreactivity of many antigens and epitopes. The negative effect of formaldehyde fixation can be reversed successfully with enzymatic digestion for some markers while not for others. Non-enzymatic epitope unmasking techniques have been recently introduced to improve the immunoreactivity of many antigens in formaldehyde fixed tissues. Heat-Induced Epitope Retrieval (HIER) in Tris-HCl buffer improves the reactivity of certain antibodies in formal-fixed tissues.

Procedure:

Supplied As:
100mM Tris-HCl buffer, pH 10.0. This is a 10X stock solution and should be diluted 10-fold with distilled water before use.

1. Place five-micron thick tissue sections on glass slides coated with poly L-lysine or APTES.

2. Deparaffinize and re-hydrate sections as usual.

3. Place slides in a Coplin jar containing 10mM Tris-HCl buffer, pH 10.0; cover with a vented plastic wrap and place the jar in a steamer for 20 minutes.

4. Take out the jar and let the sections cool in the jar for 20 minutes at room temperature. THIS STEP IS VERY CRITICAL AND SHOULD NOT BE AVOIDED.

5. Wash sections in buffer for 2x5 minutes.

6. Block endogenous peroxidase as usual.

7. Wash sections in buffer for 2x5 minutes.

8. Block non-specific sites with normal serum as usual.

9. Place optimally diluted primary antibody on the sections (incubation time and temperature for a given set of experimental conditions should be determined by the investigator).

10. Wash sections in buffer for 2x5 minutes.

11. Rest of the procedure is same as routinely performed in your laboratory.

Suggested Working Dilution:
Immunohistology: 1:10 with distilled water.

Suggested Test Size:
It is recommended that at least 75 ml of Tris-HCl buffer should be used for 12 slides.

IHC Protocol Summary