ESO wikiadmin at 08:36, 20 May 2025

2025-05-20T08:36:13Z

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 |Mounting medium
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-§   Transfer coverslips to microscope slide (mounting medium from Dako)
+=== Procedure ===
-§   Analyze at confocal microscope
+* Seed MEF cells (2.5*10^4) on 1 µg/ml FN coated coverslips

ESO wikiadmin: Created page with "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 {| class="wikitable" |+ !Materials ! |- | |PBS++ (1x PBS + 0.9mM CaCl₂ + 0.5 mM MgCl₂) |- | |Coverslips (round, 12 mm) |- | |24 well plate |- | |4% paraformaldehyd..."

2025-03-07T09:49:18Z

Created page with "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 {| class="wikitable" |+ !Materials ! |- | |PBS++ (1x PBS + 0.9mM CaCl2 + 0.5 mM MgCl2) |- | |Coverslips (round, 12 mm) |- | |24 well plate |- | |4% paraformaldehyd..."

New page

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

{| class="wikitable"
|+
!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

Immunofluorescence staining of focl adhesion proteins - Revision history

ESO wikiadmin at 08:36, 20 May 2025