Rhodamine phalloidin

Organ cultures or epithelial cells were fixed with 4% paraformaldehyde and corneas were sectioned radially. The tissue or cells were permeabilized with 0.1% v/v Triton X-100 in phosphate buffered saline (PBS) and blocked with 4% bovine serum albumin (BSA) in PBS for indirect immunofluorescence. Samples were incubated in a specific primary antibody solution overnight at 4°C, washed, and incubated in Alexa Fluor-conjugated secondary antibodies (1:100; Thermo Fisher Scientific) in 1% BSA in PBS for 1 hour at room temperature. Samples were counterstained with rhodamine phalloidin (1:50; Thermo Fisher Scientific) to visualize F-actin and mounted using VectaSHIELD with DAPI to stain nuclei (Vector Labs). Images were taken on a Zeiss LSM 700 confocal microscope (Zeiss, Thornwood, NY) using either the 40× oil or 63× oil objective. Secondary antibody controls were used to set the gain of the laser at a negative or black and all experimental samples were imaged at that gain. Pinhole size was kept at 1 Airy Unit and images were obtained using Zen Black Edition software (Zeiss, Thornwood, NY). Analysis was performed using FIJI/ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij/).

The following antibodies were used: antibody against human β1 integrin, isotype antibody control IgG2a,κ, anti-FN, anti-tensin (all from BD Biosciences), anti-human integrin-α5 nonfunction-blocking mAb11 [26 (link)], anti-human integrin-α5 inhibitory mAb16 [27 (link)], anti-human integrin-β1 inhibitory mAb13 [28 (link)], anti-human integrin-αV L230 (ATCC), anti-human FN mAb 13G12 [28 (link)], anti-β3 integrin (sc-7311; Santa Cruz Biotechnologies), anti-phospho-FAK (Fischer Scientific). Other antibodies used were from Sigma-Aldrich: anti-actin, anti-talin, anti-α-actinin, anti-vinculin and anti-paxillin. Secondary species–specific FITC-, Cy3- or AMCA-conjugated antibodies were from Jackson ImmunoResearch Laboratories. Rhodamine-phalloidin was from Fischer Scientific.
Human plasma FN was purified according to Miekka [29 (link)] and 70kD fibronectin fragment was obtained as described [30 (link)]. The 120kD fragment from plasma FN was purchased from Merck. Fibronectin-depleted FBS was obtained by the use of Gelatin-Sepharose 4B (LKB) as described by Knox [31 (link)]. Cellular fibronectin was purified according to Yamada et al. [32 (link)]. HiLyte Fluor™ 488 labeled bovine FN was purchased from Cytoskeleton Inc.

MAP gels with cells cultured for 2 days were fixed in 1% paraformaldehyde for 15 minutes at 25°C. The cultures were permeabilized in 0.1% Triton X-100 in PBS and stained using DAPI (Sigma-Aldrich) for cell nuclei and rhodamine phalloidin (Thermo Fisher) for cell actin per manufacturer’s guidelines for 1 hour. Gels were washed with PBS before z-stack imaging with a Nikon confocal. To quantify cell spreading, the z-stacks were imported into IMARIS to generate surface renders of cell actin for surface area quantification and to count nuclei.