Zen black edition software

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/).

Immunostaining of KB-VIN was performed as described previously.^{43 (link)} Briefly, KB-VIN cells were seeded on an eight-well chamber slide (Lab-Tech) for 24 h prior to treatment with compounds. Cells were treated with compound for 24 h. Concentrations of reagents were used based on their IC₅₀ used for cell cycle analysis as follows: 0.3 and 3.0 µM of 1, 20 µM 3, and 0.1% DMSO as a control. Cells were fixed in ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS) followed by permeabilization with 0.5% Triton X-100 in PBS. Fixed cells were labeled with mouse monoclonal antibody to α-tubulin (B5-1-2, Sigma) and rabbit polyclonal antibody to γ-H2AX (BETHYL Lab.) followed by FITC-conjugated antibody to mouse IgG (Sigma) and Alexa Fluor 546-conjugated antibody to rabbit IgG (Life Technologies). Nuclei were labeled with DAPI (Sigma). Fluorescence-labeled cells were observed using a confocal microscope (Zeiss, LSM700) controlled by ZEN software (Zeiss). Parameters (laser, beam splitter, band-pass filter) for confocal fluorescence imaging were used as follows: track 1 for DAPI (405 nm, 420 nm, 420−1000 nm), track 2 for FITC (488 nm, 544 nm, 490−555 nm), and track 3 for Alexa Fluor 546 (555 nm, 559 nm, 505−600 nm). Confocal images were reconstructed by stacking using ZEN (black edition) software (Zeiss). Final images were prepared by Photoshop CS6 (Adobe).

Corneal epithelial cells or organ cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min (cells) or for 30 min (organ cultures) at room temperature. Corneas were sectioned radially and immunofluorescence staining was performed. The cells or tissues were permeabilized for 5 min with 0.1% v/v Triton X-100 in PBS and blocked for 3 hr at room temperature with 4% bovine serum albumin (BSA) in PBS. Samples were incubated in primary antibody solutions overnight at 4°C, washed, and then incubated in Alexa Fluor conjugated secondary antibodies (1:100) (Thermo Fisher Scientific) in 1% BSA for 1 hr at room temperature. The samples were counterstained with rhodamine phalloidin (1:50) (Thermo Fisher Scientific) to examine F-actin. The cells or tissues were mounted using VectaSHIELD with DAPI (Vector Laboratories). Images were taken using a Zeiss LSM 700 confocal microscope (Zeiss, Thornwood, NY) with either a 40× oil or 63× oil objective. Secondary antibody controls were used to set the gain of the laser, and experimental samples were imaged at the same gain. The pinhole size was kept at 1 Airy unit across all images. Images were obtained using Zen Black Edition software (Zeiss) and analyzed with FIJI/ImageJ (NIH, Bethesda, CA).