Mouse anti rab7

Primary MN were grown on 13 mm glass cover slides, and then fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. Cover slides were then washed in PBS and permeabilized with 0.5% Triton in blocking solution containing 5% donkey serum (Jackson Laboratories) and 1 mg/mL BSA (Sigma) for 5–30 min. After three washes, cells were incubated with appropriate antibodies overnight at 4°C in blocking solution [rabbit anti-Dicer (Abcam,1:100), rabbit anti-Ago2 (Abcam,1:200), chicken anti-NFH (Abcam and Millipore, 1:1000), mouse anti-neurofilament phosphorylated (SMI31)/non-phosphorylated (SMI32) (Covance, 1:500), mouse anti-Rab7 (Abcam, 1:300), mouse anti-TrkB (BD Bioscience, 1:200), mouse anti-COX IV (1:200; Cell Signaling Technology)]. After having been washed, secondary antibodies (2 μg/mL Jackson Laboratories, ThermoFisher) in blocking solution were added for 2 h at room temperature. Phalloidin was used to visualize F-actin, and added when necessary to the secondary antibody. For axonal visualization, cells were incubated for 45 min with Alexa Fluor 594/405-conjugated phalloidin (1x, Abcam), diluted in PBS, then washed and mounted with ProLong Gold (Life Technologies). For mitochondria staining, the cells were incubated with Mitotracker Deep Red FM (100 nM, Thermo Fisher) for 30 min at 37°C, and then washed 3 times with PBS prior to fixation.

APPswe/HEK293 cells were coated coverslips in 24-well culture dishes before transfection. After transfection, cells were incubated with primary antibodies including rabbit anti-APP (1:100, Sigma), mouse anti-EEA1 (1:100, cell signaling technology), mouse anti-Rab7 (1:50, abcam), and mouse anti-LAMP1 (1:10, abcam). The images were captured using a Pannoramic MIDI (3D Histech, Hungary) equipped with a GS3-U3-51S5M-C camera (FLIR, Canada), Lumencor SOLA (Beaverton, OR), and Semrock filters (Rochester, NY). Pearson’s coefficients of colocalization were analyzed by ImageJ (NIH).

Latrunculin B (Calbiochem-Novabiochem Corp., San Diego, CA, USA) and nocodazole (Sigma-Aldrich, Saint Louis, MO, USA) were used as previously described to disrupt actin and microtubule cytoskeleton integrity, respectively [27 (link)]. Cycloheximide (Sigma-Aldrich) was used to inhibit protein synthesis in HUVECs at the concentration of 50 μg/mL.
The following primary antibodies were used: mouse monoclonal anti-CD93 (mAb 4E1) [6 (link)], rabbit anti-MMRN2 (generously provided by M. Mongiat), rabbit anti-CD93 (HPA009300, Atlas Antibodies, Bromma, Sweden), mouse anti-CD93 (MBL International Corporation, Woburn, MA, USA), rabbit anti-Giantin, mouse anti-β1 integrin (12G10), and mouse anti-Rab7 (Abcam, Cambridge, UK), rabbit anti-Moesin (Cell Signaling Technology, Danvers, MA, USA), mouse anti-Rab5 (BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-β-actin (Sigma-Aldrich), rabbit anti-CD93 (H-190), mouse anti-COPD (E-12), mouse anti-Sec31A (H-2), mouse anti-β-Adaptin (A-5), mouse anti-Rab5a (E-11), mouse anti-Rab5b (F-9), mouse anti-Rab5c (H-3), mouse anti-β1 integrin (4B7R), mouse anti-Rab11a (D-3), rabbit anti-caveolin-1 (N-20), and mouse anti-MMRN2 (H572) (Santa Cruz Biotechnology, Dallas, TX, USA). Alexa Fluor-488 and -647 phalloidin (Thermo Fisher Scientic) were used for F-actin labeling.