Evaluation of Apoptosis in Lens Epithelial Cells

In order to evaluate whether apoptosis occurred in lens epithelial cells adhering to anterior lens capsule following B-MICS surgical approach and after release of pulse energy by femtosecond laser, samples were processed for confocal immunofluorescence analysis, as previously described [13 (link), 14 (link)]. Briefly, capsules fixed with 4% were washed with PBS and underwent saturation with PBS containing 3% bovine serum albumin (BSA) for 30 minutes at room temperature. Then, samples were incubated with a rabbit anti-cleaved caspase 3 primary antibody (Cell Signaling) diluted 1 : 50 in PBS containing 3% BSA, for 1 hour at room temperature. After washing in PBS containing 3% BSA, specimens were incubated with a goat anti-rabbit Alexa546 secondary antibody (Life Technologies), diluted 1 : 200 in PBS containing 3% BSA. Samples were then washed with PBS, stained with 1 mg/ml 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 1 minute, and then mounted with antifading medium. Fluorescent samples were observed by a Nikon A1 confocal laser scanning microscope. The confocal serial sections were processed with ImageJ software to obtain three-dimensional projections and image rendering was performed using Adobe Photoshop Software [15 (link)].