Tris buffered saline (tbs)

Staining was performed, as previously described (6 (link)). The cells were fixed in 4% paraformaldehyde solution (Thermo Fisher Scientific Inc., Rockford, IL, USA) for 15 min, following which they were incubated with donkey serum blocking buffer [Tris-buffered saline containing 5% (w/v) normal donkey serum (Jackson ImmunoResearch, Hamburg, Germany)] for 30 min and the primary antibody for 60 min. Subsequently, the cells were incubated with donkey anti-mouse polyclonal immunoglobulin G (IgG) and/or donkey anti-rabbit polyclonal IgG (DyLight 488 and 594, respectively; Jackson ImmunoResearch) for 60 min at room temperature. The following antibodies were used: Mouse monoclonal anti-DSC2 (7G6; Invitrogen Life Technologies), rabbit polyclonal anti-E-cadherin (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-γ-catenin (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-DSG2 (10G11; Progen Biotechnik GmbH), mouse monoclonal anti-PKP2 (PP2/62, PP2/86, PP2/150, Progen Biotechnik GmbH), mouse monoclonal anti-pan-cytokeratin (Santa Cruz Biotechnology, Inc.) and Acti-stain™ 555 Fluorescent Phalloidin (Cytoskeleton Inc., Denver, CO, USA).

Cells were washed with ice-cold PBS and lysed with RIPA Buffer (Sigma) supplemented with Protease Inhibitors (Thermo Fisher Scientific), phosphatase inhibitor cocktail (Pierce), and 1 mM DTT (Sigma) in a 6-well plate. Lysate was then centrifuged for 10 min at 4°C at 21,000Xg. Pierce Bicinchoninic Acid Protein Assay Kit (Thermo Scientific) was used to determine protein concentration. 30 μg of protein per well was used to detect protein expression levels. Lysates were run on 4%–12% Bis-Tris gel (Invitrogen), transferred to PVDF, and blocked with 5% milk (Research Products International). Blots were stained overnight at 4°C with total AMPK (Cell Signaling Technology) and pAMPK Thr 172 (Cell Signaling Technology) antibodies in 2% bovine serum albumin (Gemini Bio). Blots were washed with Tris-Buffered Saline and stained with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch). Blots were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo). Total protein loading was assessed by BlotFastStain (G-Biosciences). Blots were imaged using the Bio-Rad ChemiDoc MP imaging system and band density was quantified using ImageJ.

Cryosections from Long–Evans rats left eye were fixed with 4% paraformaldehyde in 0.1 M PBS (Irvine Scientific) at room temperature for 20 min. These fixed cells and sections were blocked and permeabilized with a blocking solution (Tris-buffered saline (TBS), 0.3% Triton X-100 and 3% goat serum (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) for 15 min. Samples were then rinsed twice with 0.1 M TBS buffer for 15 min each time, mounted on polysine microscope slides and incubated with primary antibodies overnight at 4 °C (rhodopsin-APC, RG-opsin-APC, STEM121-FITC, DAPI-VioBlue) at concentrations determined in laboratory. Post-overnight incubation, samples were rinsed three times with TBS for 15 min. Secondary antibody (goat-derived anti-mouse and anti-rabbit, DAPI-VioBlue) staining was performed for 1 h at room temperature. Samples were then washed one last time with TBS before being mounted on poly-l-lysine microscope slide with low viscosity slide mounting medium. Digital images were obtained with an epifluorescence confocal microscope (Leica SP8) using 20× objective.