Cells grown on glass coverslips in complete medium in 6-well plates were treated with alcohol (0.2%–43mM) for the indicated times in three separate experiments. Cells were then washed with PBS, fixed/permeabilized with paraformaldhyde/triton x-100, and stained with primary and fluorescent secondary Abs as described (Forsyth et al., 2007). For the TACE/ZO-1 images (Fig. 6C) (20 × 1μM z-stacks for 3D) were taken. All images were taken using a Zeiss Axiovert 100 microscope with Axiovision software (Carl Zeiss Inc, Thornwood, NY) using an oil immersion 40x objective as described (Forsyth et al., 2007). The plane of focus selected was the one that revealed the greatest overall clarity for any given image. Quantitative analysis of immunofluorescent staining for vimentin (Fig. 1) was performed with Image J software (NIH) using blinded selection and analysis to prevent bias. Three separate images from three separate experiments were analyzed by determining immunofluorescent intensity in five areas from each of the three images (thus N=15) for each condition. Background values were subtracted using images for cells treated only with secondary antibodies. Means were compared for control and alcohol treated cells for each cell type by t-test using SPSS with p≤.05 being significant.
