Immuno fluorescence on adherent cells

1. PBSM (1x PBS + 0.5 mM MgCl₂)

2. PFA
   

3. Glycine

4. Triton-X-100

5. BSA
   

6. primary antobody

7. PBT (1x PBS+ 1% Triton)
   

8. DAPI

9. Coverslips

10. Glass slides

11. Prolong Gold

12. Nail polish

1. Wash with PBSM (=PBS + 5mM MgCl2) (3 quick rinses)

2. Fix cells 10 min RT in 4% Paraformaldehyde in PBSM

3. Wash with 0.1M Glycine/PBSM 10 min @ RT (quenching)

4. Wash with PBSM 2 x 10 min @ RT (cells can be left overnight @ 4°C at this point).

5. Permeabilize cells 5 min with PBS supplemented w/ 0.2% triton x100. (0.2% PBT)

6. Blocking: incubate for 30' in 0.1% PBT with 3% BSA.

7. Place coverslip over 50μl of primary Ab diluted in 0.1% PBT with 3% BSA in a humid chamber for 2h at RT.

8. Wash 3x5' with PBT 0.1%.

9. Place coverslip over 50μl of second Ab  diluted in 0.1% PBT with 3% BSA in a humid chamber for 20-30 min at RT.

10. Wash 3x5' with PBT 0.1%.

11. Wash 5min in PBS.

12. Mount coverslip onto a slide using mounting medium (prolong gold+DAPI).

Note: Always have negative control of the secondary antibody (without the primary antibody)