Mouse anti beta 3 tubulin

The following antibodies were used in this study: rat anti-mouse LAMP1 (1D4B) from BD Biosciences, goat anti-mouse Cathepsin D (C-20) from Santa Cruz and mouse anti-beta III tubulin from Promega. Rabbit anti-subunit c of mitochondrial ATP synthase (SCMAS) antibodies [32 (link)] were a gift from Dr. Elizabeth F. Neufeld (David Geffen School of Medicine, University of California, Los Angeles, CA). Rabbit anti-TMEM106B antibodies were generated against the cytosolic domain of human TMEM106B as previously described [6 (link)].

For whole-gel differential immunostaining, cultures were fixated for 2 h in 4% paraformaldehyde (PFA) solution in PBS, and then permeabilized and blocked with 0.3–1% Triton and 4% fetal bovine serum (FBS) solution in PBS (4 h). The cultures were then washed with PBS, and incubated (overnight, 4°C) with the following primary antibodies: Mouse-Anti beta III tubulin (1:400, Promega), a neuronal cell marker, and Rabbit-Anti S100 (1:200, Sigma), a marker of glial cells. Cultures were then washed with PBS and exposed (4 h to overnight) to CY3-conjugated goat-anti-mouse IgG (1:100, Jackson), Dylight 488-conjugated goat-anti-rabbit IgG (1:100, Jackson), and DAPI (6 nM, Sigma), designed to stain the nuclei of all cells and incubated for at least 4 h, before being washed again with PBS. Cells were imaged using a confocal microscope (Zeiss, LSM 700).