Immunofluorescence staining of focl adhesion proteins
| Materials | |
|---|---|
| PBS++ (1x PBS + 0.9mM CaCl2 + 0.5 mM MgCl2) | |
| Coverslips (round, 12 mm) | |
| 24 well plate | |
| 4% paraformaldehyde (PFA) in PBS | |
| Permeabilization/fixation solution (4% PFA/PBS + 0.1% Triton-X-100) | |
| Blocking solution (10% heat inactivated CS in PBS) | |
| Mounting medium |
Procedure
- Seed MEF cells (2.5*10^4) on 1 µg/ml FN coated coverslips
- Optional: treat cells with inhibitor/stimulus etc. for indicated timepoints
- Wash cells 1 x with PBS++
- 1st fixation step with 350 µl of 4% PFA/PBS + 0.1% Triton-X-100 for 5 min at RT (in the dark if your sample expresses fluorescent proteins)
- Remove 1st fixation and add 350 µl of 4% PFA/PBS. Incubate for 15 min at RT in the dark
- Wash 3x with PBS++
- Add 300µl blocking solution and incubate for 10 min in the dark
- Apply 1st antibody at appropriate dilution (e.g. phosphotyrosine pY72 1:100) in blocking solution. Apply to the center of the coverslip, make sure that coverslip does not touch wall of the well.
- Incubate for 1h at RT in humidified chamber
- Wash 3x with PBS++
- Add 300 µl blocking solution for 10 min in the dark.
- Apply 2nd antibody of choice in appropriate dilution (1:200). May be combined with phalloidin staining (1:100). Incubate for 30 min at RT in humidified chamber (dark)
- Wash 3x with PBS++
- Transfer coverslips to microscope slide (mounting medium from Dako)
- Analyze at confocal microscope