Western Blot Analysis of Retinal Proteins

Western Blotting was performed as previously described [73 (link)]. Briefly, protein separation from retinas (N = 5) were performed on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). This was followed by protein electrotransfer to polyvinylidene difluoride (PVDF) membrane (IPFL10100; Merck Millipore, Milan, Italy). Blocking of membranes was performed with a 5% non-fat dry milk/Tris Buffered Saline (TBS) solution for 1 h. Then, membranes were incubated overnight at 4 °C with the following primary antibodies, all diluted in 3% blocking solution: anti-Iba1 (3 µg/mL; ab5076; Abcam; PLC., Cambridge, UK); anti-LC3B (1 µg/mL; ab48394; Abcam; PLC., Cambridge, UK), and β-actin (1:1000; sc47778; Santa Cruz Biotechnology, Dallas, TX, USA). Blots were then incubated with horseradish peroxidase-conjugated secondary anti-rabbit, anti-goat, and anti-mouse antibodies (all 1:10000; respectively, sc-2004, sc-2020, and sc-2005; Santa Cruz Biotechnology, Dallas, TX, USA), for 1 h at room temperature. Iba1 and LC3B immunoreactive bands were detected by using an enhanced chemiluminescence system (35055; Thermo Fisher, Waltham, MA, USA). Then, these were quantized with VisionWorks Life Science Image Acquisition and Analysis software (UVP, Upland, CA, USA), normalized with β-actin protein levels and expressed as densitometric units (DU).