Immunofluorescence Insulin BrdU
Reagent:- Guinea pig anti-swine insulin antibody from Dako #A0564.
- Rat anti-BrdU antibody from Serotec #MCA2060.
- Donkey anti-Guinea pig IgG, FITC conjugated from Jackson Immunoresearch #706-095-148.
- Donkey anti-rat IgG, Cy3 conjugated from Jackson Immunoresearch #712-165-150.
- Antigen retrieval solution (10X) from Vector laboratories #H-3300.
- Humid chamber, black, from Newcomer #68432A.
- 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 Glut2 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. Antigen retrieval. Put the slides in glass Coplin jar. Add 1 X antigen retrieval solution and heat in microwave for 1 min until boil. Cool down slowly to RT. Wash with 1 X PBS for 3 times, 5 min each.
4. 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-BrdU antibody 1:50 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.
5. Wash with 1 X PBS for 3 times, 5 min each.
6. Secondary antobody incubation. Prepare the secondary antibody mixer by diluting anti-guinea pig IgG-FITC of 1:250 and anti-rat IgG-Cy3 of 1:250 using 1 X PBS. Add to the tissue sections and store in the humid chamber for 1 hr at RT.
7. Wash with 1 X PBS for 3 times, 5 min each.
8. 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.
9. Wash with 1 X PBS for 3 times, 5 min each.
10. Drain the liquid from the tissue sections and add a drop of anti-fade and slowly cover with coverslip.
11. Seal the coverslip using transparent nail polish.
12. Acquire image immediatedly or store at -20C for long term.