Immunofluorescence insulin MafA
Reagents:
- Guinea pig anti-swine insulin antibody from Dako #A0564.
- Rabbit anti-MafA antibody from Bethyl labs #A300-611A.
- Donkey anti-Guinea pig IgG, FITC conjugated from Jackson Immunoresearch #706-095-148.
- Donkey anti-rabbit IgG, Cy3 conjugated from Jackson Immunoresearch #711-165-152.
- Humid chamber, black, from Newcomer #68432A.
- Paraformaldehyde (16%) from EMS #RT15711.
- Anti-fade from Invitrogen #P36934.
- Transparent nail polish from CVS.
- Primary antibody dilution buffer: 1% BSA, 0.1% cold fish skin gelatin, 0.05% sodium azide, 1 X PBS. (Recipe from IHCworld.com)
This protocol has been successively used for immunofluorescence staining for insulin and MafA simutaneously in mouse pancreas tissues.
The key for MafA staining is the fixation procedure.
1. Harvest the pancreas tissue into cold 4% paraformaldehyde/PBS solution and incubate for 2 hr at 4C.
2. Rinse twice in cold 1 X PBS, 5 min each.
3. Transfer the tissue to cold and transfer to 30% sucrose/PBS and keep at 4C overnight.
4. Transfer to 50%OCT/50% sucrose (30%) solution and let it sink for 15-30 min at RT. Shake every 2-3 min.
5. Transfer to 100% OCT and keep at RT for 30 min.
6. Transfer to new 100% OCT and freeze on dry ice.
7. Store at -80C until processing. Store the slides with sections at -80C.
8. When staining, take out from -80C and let the slides warm to RT for 30 min.
9. Wash in 1 X PBS, twice, 5 min each.
10. 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-MafA antibody 1:1000 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.
11. Wash with 1 X PBS for 3 times, 5 min each.
12. 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.
13. Wash with 1 X PBS for 3 times, 5 min each.
14. 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.
15. Wash with 1 X PBS for 3 times, 5 min each.
16. Drain the liquid from the tissue sections and add a drop of anti-fade and slowly cover with coverslip.
17. Seal the coverslip using transparent nail polish.
18. Acquire image immediatedly or store at -20C for long term.