Vps35 Mutant Mice Characterization

Vps35 mutant mice have been described previously [9 (link),10 (link),14 (link)], which were backcrossed with C57BL/6 mice for more than 10 generations. Mice were maintained on a standard rodent diet. Vps35 mutations were confirmed by genotyping using PCR and Western blot analysis. All experimental procedures were approved by the Animal Subjects Committee at the Georgia Regents University according to US National Institutes of Health guidelines.
Rabbit polyclonal anti-VPS35 antibody was generated using the antigen of GST-VPS35D1 fusion protein as described previously [9 (link),10 (link),14 (link)]. Rabbit polyclonal antibodies, including anti- ß-galactosidase (Cappel), anti-VPS26 (Abcam),anti-calretinin (Swant), and anti-melanopsin (Advanced Targeting Systems) antibodies, were purchased. Mouse monoclonal antibodies, including anti-neuronal class III ß-Tubulin (Tuj1,Convance), anti-neurofilament (DSHB), anti-calbindin D28k (Swant), anti-glial fibrillary acidic protein (GFAP, Chemicon), anti-rhodopsin (Abcam) and anti-myelin basic protein (MBP, Chemicon) antibodies, were also purchased. In addition, the chicken polyclonal anti- ß-galactosidase antibody (Abcam) and goat polyclonal anti-IBA1 antibody (Abcam) were used. Secondary antibodies were purchased from Jackson Immuno Research Laboratories, Inc. Other chemicals and reagents used in this study were of analytical grade.