Immunofluorescence Insulin TUNEL

Reagents

This protocol has been successively used for immunofluorescence staining for apoptotic beta cells in paraffin embedded mouse pancreas tissues.

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

2. Deparaffinize the sections by the following solutions.

3. Antigen retrieval. Use PAP pen makes a circle for each tissue section which can prevent liquid flow outside. Prepare protease K solution of 20 ug/ml. Add protease K solution to cover each sample, ~150 ul per section. Incubate at 37C for 15 min.

4. Wash the sections using 1 X PBS, 3 times, 5 min each.

5. TUNEL staining. Prepare the enzyme mix by adding X ul enzyme to substrate vial. Add the mix to tissue sections and incubate at 37C for 1 hr.

6. Stop the reaction by washing out the enzyme mix and wash with 1 X PBS for 3 times, 5 min each.

7. Primary antibody incubation. To co-stain the beta cells, prepare the primary antibody mixer by diluting anti-insulin antibody of 1:500 using primary antibody dilution buffer. Add water to the humid chamber and put tissue slides into it. Add the primary antibody mixer to tissues sections and store at 4C for overnight.

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

9. Secondary antobody incubation. Prepare the secondary antibody mixer by diluting anti-guinea pig IgG-FITC of 1:250 using 1 X PBS. Add to the tissue sections and hybridize in the humid chamber for 1 hr at RT.

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

11. Prepare DAPI working solution by adding 0.5 ul DAPI stock solution of 10 mg/ml to 50 ml 1 X PBS. Incubate with tissue sections for 5 min at RT.

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

13. Drain the liquid from the tissue sections and add a drop of anti-fade and slowly cover with coverslip.

14. Seal the coverslip using transparent nail polish.

15. Acquire image immediatedly or store at -20C for long term.


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