Immunohistochemistry Protocol

Immunohistochemistry Protocol for Polyclonal Antibody

Primary Antibody

1. Take 4 micron paraffin sections down to water

2. Block endogenous peroxidase with 3% Hydrogen Peroxide for a total of 15 minutes incubation

3. Wash the sample in running water

4. HEIR Method

In a plastic coplin jar, immerse slides in 10mM of citrate buffer, pH of 6.0. The coplin jar should then be placed in a microwaveable pressure cooker surrounded by hot water. Microwave the sample for a total of 20 minutes.

5. Wash slides in water and then rinse in T.B.S. pH 7.6

6. Block with a concentrated blocking reagent for 10 minutes

7. Drain blocking reagent and apply antibody at optimized dilution concentration. Let incubate for 1 hour at room temperature

8. Wash with T.B.S. two times at 1 minute each

9. Drain T.B.S. and apply LINK at a dilution of 1:4 for 30 minutes at room temperature

10. Wash again with T.B.S. twice at 1 minute each

11. Drain T.B.S. and apply USA PO dilution 1:4 and incubate at 30 minutes at room temperature

12. Wash in T.B.S. twice for 1 minute each, then 1 at 5 minutes

13. Develop with a chromogen AEC for 10 minutes

14. Wash with water

15. Counter stain in Mayer's Haematoxylin

16. Cover sections with Crystal Mount