Antibody and cell staining reagents
Nestin (clone Rat 401, 2 μg/ml), Lamp 1 (clone 1D4B, 6 μg/ml), and Lamp 2 (clone Abl-93; 6 μg/ml) monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, maintained by The University of Iowa, Department of Biological Sciences. Batp1 (1 μg/ml) was previously described [12]. Anti-subunit c antibody (0.7 μg/ml) was kindly provided by Dr. Kominami (Juntendo University, Tokyo, Japan). Additional antibodies were as follows: GFAP, 1:2000 (DAKO Corporation); calbindin, 1:5000 (Sigma); NeuN, 10 μg/ml (Chemicon); SV40 T antigen (Pab 101), 2 μg/ml (Santa Cruz Biotechnology); cathepsin D (C-20), 2 μg/ml (Santa Cruz Biotechnology); cytochrome c oxidase subunit IV (cox4), 1:1000 (BD Biosciences Clontech); PDI (H-160), 2 μg/ml (Santa Cruz Biotechnology); GM130, 1 μg/ml (BD Transduction Labs); α-tubulin, 1:15,000 (Sigma); grp75, 1:200 (Stressgen); early endosome antigen-1 (EEA1), 2 μg/ml (Santa Cruz Biotechnology); rab 7, 4 μg/ml (Santa Cruz Biotechnology). All fluorescent secondary antibodies were obtained from Jackson ImmunoResearch Laboratories and HRP-conjugated secondary antibodies were obtained from Amersham Biosciences. Additional cell markers were as follows: VVL-biotin, 1:2000 (Vector Laboratories), 10,000 MW dextran-FITC, 1 mg/ml and Lysotracker Red DND-99, 500 nM (Molecular Probes).