View source for Immunofluorescence staining of focl adhesion proteins

Materials

PBS++ (1x PBS + 0.9mM CaCl<sub>2</sub> + 0.5 mM MgCl<sub>2</sub>)

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

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!Materials
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|PBS++ (1x PBS + 0.9mM CaCl<sub>2</sub> + 0.5 mM MgCl<sub>2</sub>)
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|Coverslips (round, 12 mm)
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|24 well plate
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|4% paraformaldehyde (PFA) in PBS
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|Permeabilization/fixation solution (4% PFA/PBS + 0.1% Triton-X-100)
|-
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|Blocking solution (10% heat inactivated CS in PBS)
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|Mounting medium
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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