Polyclonal rabbit anti lamp1

The mice were sacrificed by decapitation and the brains were quickly removed. The left halves of the brains were fixed in 4% paraformaldehyde and embedded in paraffin, while the right halves were quickly frozen and stored at −80°C for biochemical analysis. Immunohistochemistry was performed according to methods described previously.[19 (link)] For 6E10 staining, which detects Aβ, the blocking step was preceded by treatment with 70% formic acid for 10 min. The following primary antibodies were used: 6E10 (monoclonal mouse anti-Aβ1-16 1:1000, Biolegend, USA), polyclonal rabbit anti-Rab5 (1:300, Abcam, USA), polyclonal goat anti-Rab7 (1:100, Santa Cruz, USA), polyclonal rabbit anti-LC3B (1:200, Cell Signaling Technology, USA), polyclonal rabbit anti-Lamp1 (1:400, Abcam), polyclonal goat anti-Beclin1 (1:100, Santa Cruz), polyclonal rabbit anti-UVRAG (1:400, Millipore, USA), polyclonal rabbit anti-Rubicon (1:50, LifeSpan Biosciences, USA), Alexa Fluor-conjugated antibodies (Molecular Probes, USA), and HRP-conjugated antibodies (chromogenic). Nuclei were labeled with DAPI or hematoxylin. Images were acquired using a confocal microscope (Fluoview10, Olympus, Tokyo, Japan) or an epifluorescence microscope (Olympus).

RGCs were loaded with radio immunoprecipitation assay buffer. Lysates were collected by centrifugation at 12 000 rpm for 5 min at 4 °C. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for western blotting. Membranes were blocked for 1 h at RT in 5% nonfat dry milk, then incubated with polyclonal rabbit anti-parkin (1 : 1000; Abcam, USA), monoclonal rabbit anti-Bax (1 : 1000; Abcam), polyclonal rabbit anti-Bcl-2 (1 : 500; Abcam), monoclonal rabbit anti-LC3 (1 : 2000; Abcam), polyclonal rabbit anti-LAMP1 (1 : 1000; Abcam), polyclonal rabbit anti-optineurin (1 : 200; Abcam) and polyclonal rabbit anti-GAPDH (1 : 2000; Weiao Biotechnology Co, Shanghai, China) in primary antibody dilution (Weiao Biotechnology Co) at 4 °C overnight. After washing with TBST, membranes were incubated with secondary antibodies (1 : 2000; Jackson). Blots were visualized using enhanced chemiluminescence reagents (Weiao Biotechnology Co). Chemiluminescent images were captured on X-ray film using a developing and fixing solution in a dark room and at last analyzed with Image J (National Institutes of Health, USA).