Rabbit anti akt

C2C12 myoblasts were seeded on gelatin coated coverslips and differentiated as previously described. Lysotracker 75 nM was added for 45 min when specified. After two washes with PBS, cells were fixed with 4% paraformaldehyde for 10 min. After three PBS washes, cells were permeabilized with 0.1% Triton X-100 and washed thrice with PBS. Coverslips were incubated with blocking solution (5% Fetal Bovine Serum, 1% Albumine from Bovine Serum and 0.02% sodium azide) overnight at 4°C. Subsequently, cells were incubated for 2 h at RT in a hydration chamber with the following primary antibodies: mouse anti-Aβ 6E10 1:200, rabbit anti-oligomers A11 1:200 (Invitrogen), mouse anti-phospho-Akt Ser473 1:100, rabbit anti-Akt 1:100 and mouse anti-clathrin heavy chain 1:200 (BD Biosciences). Then, cells were washed thrice with blocking buffer and incubated with Alexa Fluor 488 or 555 goat anti-mouse antibodies (Invitrogen) 1:1000 for 1 h at RT. When specified, cells were incubated with 1μM Topro-3 for 15 min, washed with PBS and mounted with Mowiol. Digital images were taken at RT with a Leica TCS SP confocal microscope with ×40 and ×63 objective and analyzed with Leica confocal software (Heidelberg, Germany) and Image J software.