Technical Reference #1833

1833.

Focal adhesions are sites of integrin extension Janet A. Askari; Christopher J. Tynan; Stephen E.D. Webb; Marisa L. Martin-Fernandez; Christoph Ballestrem; and Martin J. Humphries, University of Manchester - Wellcome Trust Centre for Cell-Matrix Research, Journal of Cell Biology, 188(1833), (2010)
Link To Paper

Materials & Methods:
Immunofluorescence - Cells were plated onto glass-bottom dishes (MatTek) precoated with ligand and allowed to spread for 60 min in serum-free culture medium. Transiently transfected GD25 cells were used 24–36 h after transfection. Cells were fixed with 3% (wt/vol) paraformaldehyde for 15 min permeabilized for 5 min with 0.5% (wt/vol) Triton X-100 (Sigma-Aldrich) and subsequently incubated for 45 min with the appropriate Alexa Fluor antibody conjugate. After washing in PBS cells were imaged at room temperature using a Delta- Vision system (Applied Precision) comprising a widefield inverted microscope (model IX-70; Olympus) with a 100x/1.35 UPLAN APO objective. Images were captured using a CCD camera (model CH350; Photometrics) and Softworx analysis software (Applied Precision). Subsequent image analysis and processing were performed using ImageJ and Adobe Photoshop software. For assays using CRRETAWAC-IgG as ligand the plates were coated with 100 μg/ml of the peptide ligand and blocked with 10 mg/ml heat-denatured bovine serum albumin (Sigma-Aldrich) before addition of cells.

Microscopic Technique
Immunofluorescence

Cell Type(s)
GD25 cells