Polyclonal rabbit anti uvrag

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).

Western blotting analysis of the entire right hemispheres or dissected cortex/hippocampi of the right hemispheres was executed as described before.[19 (link)] Polyvinylidene difluoride membranes were incubated overnight with primary antibodies, and then HRP-conjugated secondary antibodies (Jackson ImmunoResearch, USA) were applied, and chemiluminescence (Supersignal West Pico, Thermo Fisher Scientific, USA) was used to visualize the luminescence. The following primary antibodies were used for immunoblotting: polyclonal rabbit anti-Rab5 (1:1000, Abcam), monoclonal mouse anti-Rab7 (clone Rab7-117, 1:1000, Sigma-Aldrich, USA), polyclonal rabbit anti-LC3B (1:1000, Cell Signaling), polyclonal goat anti-Lamp2 (1:200, Santa Cruz), polyclonal rabbit anti-UVRAG (1:2000, Millipore), monoclonal mouse anti-Beclin1 (1:500, BD Biosciences, USA), and β-actin (clone AC-74, 1:10,000, Sigma-Aldrich).