Immunofluorescence 3T3-L1 adipocytes
Reagent:
- Rabbit anti-mouse adiponectin antibody, crude serum.
- Donkey anti-rabbit IgG, Cy3 conjugated from Jackson Immunoresearch #711-165-152.
- Humid chamber, black, from Newcomer #68432A.
- Anti-fade from Invitrogen #P36934.
- Transparent nail polish from CVS.
- Paraformaldehyde (16%) from EMS #RT15711.
- 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 adiponectin in 3T3-L1 adipocytes in vitro.
1. Replate day 4 adipocytes in 6-well plate. Sterilize 18X18 cover slips with 75% ethanol and flame, and put into the 6-well plate. Cells in one 10-cm plate can be plated to one 6-well plate.2. Allow the cells to recover for 24 hrs.
3. Wash the cells with 1 X PBS for 5 min.
4. Fix the cells in 4% paraformaldehyde (dilute in 1 X PBS) for 30 min at RT.
5. Wash the cells with 1 X PBS for 2 times, 5 min each.
6. Block unreacted groups by incubating with 50 mM NH4Cl for 5 min at RT.
7. Rinse the cells in 1 X PBS.
8. Permeablize the cells with 0.1% Triton X-100 in 1 X PBS for 5 min at RT.
9. Wash the cells with 1 X PBS for 2 times, 5 min each.
10. Primary antibody diluted in the primary antibidy dilution buffer of 1:500 and incubate with the cells for overnight in the humid chamber.
11. Wash cells with 1 X PBS for 2 times, 5 min each.
12. Incubate with secondary antibody diluted in 1 X PBS of 1:500 at RT for 1 hr.
13. Wash cells with 1 X PBS for 3 times, 5 min each.
14. Count stain with DAPI for 10 min in PBS at RT.
15. Wash cells with 1 X PBS for 2 times, 5 min each.
16. Mount cells with abtifade mounting media and invert onto glass slides.
17. Acquire picture or store at -20C until imaging.