Immunofluorescence 3T3-L1 adipocytes

Reagent:

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.


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