Immunohistochemistry Glucagon

Reagents

This protocol has been successively used for immunohistochemistry staining of insulin in paraffin embedded mouse pancreas sections.

1. Bake the sections in 60C for 30 min. I use the Isothermo hybridyzation incubator.

2. Deparaffinize the sections by the following solutions.

3. Inactivate the endogenous peroxidase activity. Use PAP pen makes a circle for each tissue section which can prevent liquid flow outside. Incubate with peroxidase blocking solution for 10 min at RT.

4. Wash the sections using 1 X PBS, 2 times, 5 min each.

5. Primary antibody. Use 1:250 dilution of glucagon antibody diluted in primary antibody dilution buffer. Incubate at 4C for overnight using the humid chamber.

6. Wash with 1 X PBS for 3 times, 5 min each.

7. Apply biotinylated secondary antibody as 1:500 in 1 X PBS. Incubate at RT for 1 hr.

8. Wash with 1 X PBS for 3 times, 5 min each.

9. Apply strepavidin/HRP as 1:200 in 1 X PBS. Incubate at RT for 1 hr.

10. Wash with 1 X PBS for 3 times, 5 min each.

11. Apply substrate. Add 1 drop of DAB chromogen to 1 ml substrate buffer and mix. Observe under microscope. When color develops, stop the reaction by immerging slides in H2O. Usually, 30 sec are enough.

12. Counterstaining with 70% Hematoxilin for 30 sec and wash with H2O.

13. Dehydration.

14. Mount the sections with VectaMount and coverslip.


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