*2.2. Immunofluorescence Microscopy*

Eyes were processed for standard para ffin embedding and 4 μm sections were prepared. Rodent Decloaker (Biocare Medical LLC, Concord, CA, USA Catalog# RD913L) was used to unmask antigens and non-specific binding was blocked with 10% normal goa<sup>t</sup> sera and 5% BSA for 1 h at room temperature. Sections received either mouse monoclonal anti-Beclin1 (BD Transduction Lab, San Jose, CA, USA, Cat#612112), or rabbit polyclonal antibodies against Atg7, Atg9 and LC3 (provided by Dr. Dunn, Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL, USA), diluted in phosphate bu ffered saline (PBS) with 1% normal goa<sup>t</sup> sera plus 1% bovine serum albumin (BSA) (Beclin1 1:20; Atg7- 1:300; Atg9 and LC3 - 1:100). After washing, sections were incubated with an appropriate FITC-conjugated secondary antibody for 1 h. In some sections, colocalization of autophagy proteins within the retinal vasculature was confirmed by dual staining with the endothelial cell marker, TRITC-agglutinin (Vector Labs, Burlingame, CA, USA, Cat#RCA120). Sections were covered with Vectashield mounting medium/DAPI (Vector Labs, Inc.). Sections were viewed using a Zeiss Axioplan 2 Upright Fluorescence Microscope with Qimage/QCapture software Version 8 (QImaging Corporate, Surrey, British Columbia, Canada). Omission of the primary antibody was the baseline control and all the fluorescence photographs were obtained under the same scaled conditions.
