Immunofluorescence Insulin Glucagon

Reagent:

This protocol has been successively used for immunofluorescence staining for insulin and glucagon simutaneously 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. Primary antibody incubation. Use PAP pen makes a circle for each tissue section which can prevent liquid flow outside. Calculate the volume required. For each section, ~150 ul solution is sufficient. Prepare the primary antibody mixer by diluting anti-insulin antibody of 1:500 and anti-glucagon antibody 1:250 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.

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

5. Secondary antobody incubation. Prepare the secondary antibody mixer by diluting anti-guinea pig IgG-FITC of 1:250 and anti-rabbit IgG-Cy3 of 1:500 using 1 X PBS. Add to the tissue sections and store in the humid chamber for 1 hr at RT.

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

7. 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.

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

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

10. Seal the coverslip using transparent nail polish.

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


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