Atg 5 antibody

Knee joint serial sections (4-μm thick) were first deparaffinized in Pro-Par Clearant (Anatech) and rehydrated in a series of graded ethanol and water. Sections were blocked with 5% serum for 30 minutes at room temperature (RT) and incubated with ATG-5 antibody (1:500 dilution, Novus Biologicals. Littleton CO. NB110-53818), LC3-antibody (1:600 dilution); MBL International (Woburn, MA) PM036), and poly(ADP-ribose) polymerase (PARP) p85 (1:60 dilution, G7341, Promega, Madison, WI) overnight at 4°C. After washing with PBS, sections were incubated with biotinylated goat anti-rabbit secondary antibody for 30 minutes at RT and then with Vectastain ABC-AP alkaline phosphatase (Vector Laboratories) for 30 minutes at RT. Slides were washed and developed in alkaline phosphatase substrate for 10 to 15 minutes. Sections were dehydrated in graded ethanol and cleared in Pro-Par Clearant (Anatech), and mounted with a coverslip.

Sections from paraffin-embedded joints were first deparaffinized in the xylene substitute Pro-Par Clearant (Anatech) and rehydrated in graded ethanol and water. Slides were cooled for 20 minutes at room temperature after antigen unmasking. After washing with PBS, sections were blocked with 5% serum for 30 minutes at room temperature. Atg5 antibody (Novus Biologicals, Littleton, CO, 1:500 dilution), LC3 antibody (MBL International, Woburn, MA, 1:1000) and PARP p85 (Promega, Madison, WI) were applied and incubated overnight at 4°C. After washing with PBS, sections were incubated with biotinylated goat anti-rabbit secondary antibody for 30 minutes at room temperature, and then incubated using Vectastain ABC-AP alkaline phosphatase (Vector Laboratories, Burlingame, CA) for 30 minutes. Specificity controls were obtained by replacing the primary antibody with non-immune rabbit immunoglobulin. Slides were washed, and sections were incubated with alkaline phosphatase substrate for 20–30 minutes.