Phalloidin alexafluor 546

Embryonic head sections or adult lens sections were labeled with anti-Aqp0a or anti-Aqp0b antibodies (1:500) overnight at 4°C. Following PBS washes, AlexaFluor 488 goat anti-rabbit secondary antibody (1:500; Thermo Fisher Scientific) was applied for 2 hours at RT. Lens plasma membranes were labeled with wheat germ agglutinin AlexaFluor 594 (1:50; Life Technologies, Grand Island, NY, USA) and DAPI (1:1000) for 2 hours at RT or overnight at 4°C. Whole permeabilized lenses were incubated in AlexaFluor Phalloidin-546 (1:200; Thermo Fisher Scientific) and DAPI (1:1000). Images were acquired with a confocal microscope and imaging software (Nikon Eclipse Ti-E with NIS-Elements AR; Nikon Corp., Tokyo, Japan). Images were viewed and compiled using a commercial viewer (NIS-Elements, version 4.2; Nikon Instruments, Melville, NY, USA) and ImageJ, version 1.51n (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and a raster graphics editor (Adobe Photoshop CS5, version 12.0; Adobe Systems, San Jose, CA, USA).

Cells were distributed onto coverslips in a multiwell plate and incubated overnight before a final concentration of 10 μM of compound was added. The cells were then incubated for 2 days, fixed with 4% paraformaldehyde (Sigma-Aldrich Co., St Louis, MO, USA) for 20 minutes at room temperature, and washed twice with phosphate-buffered saline (PBS; Thermo Fisher Scientific) with 0.1% Tween 20. For blocking, cells were further washed twice (10 minutes each wash) with PBS and incubated with 10% normal donkey serum for 30 minutes at room temperature. After incubation, the blocking solution was replaced with primary antibody solution (antiacetylated tubulin; Sigma-Aldrich Co., 1:500 dilution in blocking solution) and incubated overnight at 4°C. Post incubation, the coverslips were washed three times (30 minutes each wash) with PBS and hybridized with secondary antibody linked with Alexa 488 fluoropore (1:500 dilution; Thermo Fisher Scientific), Alexa Fluor Phalloidin 546 (Thermo Fisher Scientific), and 4′,6-diamidino-2-phenylindole (DAPI) for 1 hour at room temperature. Following this incubation, coverslips were washed three times (30 minutes each wash) with PBS, mounted, and imaged. Images were processed and quantified using ImageJ software (Version 1.49).