Quantification of Optic Nerve Proteins

Animals, deeply anesthetized, were sacrificed and perfused with normal saline [6 (link),9 (link),21 (link),22 (link),23 (link)]. Subsequently, the optic nerve, chiasm, and tract were dissected and lysed in cell lysis buffer (Cell lytic M lysis buffer; Sigma-Aldrich). After determining the protein concentration using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), 6 µg of protein extract underwent separation by 15% SDS-PAGE, followed by transfer to a PVDF membrane (Millipore, Darmstadt, Germany). Immunostaining was performed using either anti-gal-3 antibody (1:500; Santacruz Biotechnologies) or anti-GAPDH antibody (1:5000; Sigma-Aldrich). HRP-conjugated secondary antibodies (GE Healthcare Systems, Sunnyvale, CA, USA) were applied, and the chemiluminescence assay (ECL) from GE Healthcare Systems was used. Chemiluminescence signals were detected using a Luminoimage Analyzer system (ChemiDoc Touch MP; Bio-Rad).