Sheep anti mouse immunoglobulin g

Brains were lysed in a protein extraction buffer containing 50 mM Tris (pH 7.4), 2% SDS, and complete protease inhibitor cocktail (Roche, Mannheim, Germany) and homogenized by sonication. The insoluble debris was removed by centrifugation, and the protein concentration of the supernatant was measured by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). Western blot analysis was performed as described previously [20 (link)]. Twenty microgram protein was loaded per lane on SDS polyacrylamide gels (ExcelGel SDS, gradient 8–18, Amersham Biosciences) on a horizontal electrophoresis system (Amersham Biosciences) and thereafter blotted onto a nitrocellulose membrane. For the detection of specific antigens, the following first step antibodies were used: rabbit polyclonal anti-p62 (BML-PW9860-0100, Enzo Life Sciences, NY, dilution 1:2000), rabbit anti-Atg7 (Sigma, 1:1000), and mouse anti-GAPDH (HyTest Ltd., Finland, 1:2000). As secondary antibodies, goat anti-rabbit immunoglobulin G (IgG) (Bio-Rad Laboratories, CA) and sheep anti-mouse immunoglobulin G (GE Healthcare, UK) antibodies conjugated to horseradish peroxidase were used at a dilution of 1:10000. The bands were revealed with enhanced chemiluminescence reagent (ThermoFisher Scientific).