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