Immpress ap anti rabbit igg polymer detection reagent

Explants were fixed in 10% zinc-buffered formalin (Z-Fix; Anatech) for 24-hours and embedded in paraffin. Paraffin-embedded samples were deparaffinized in the xylene substitute Pro-Par Clearant (Anatech) and rehydrated in graded ethanol and water. After washing with TBS, sections were blocked with 2.5% horse serum for 1 hour at room temperature and then incubated with rabbit anti-cleaved poly (ADP-ribose) polymerase (PARP) polyclonal antibody (Promega; dilution 1:50) overnight at 4°C. After washing with TBS, sections were incubated with ImmPRESS-AP Anti-Rabbit IgG Polymer Detection Reagent (Vector Laboratories) for 30 min following manufacturer's instructions, counterstained with hematoxylin (Thermo Scientific), dehydrated and mounted. Positive and total cells were counted in two different sections per explant, and results are reported as percentage of PARP positive cells.

Paraffin-embedded human and mouse samples were deparaffinized and rehydrated as described elsewhere (15 (link)). 5-μm thick sections were blocked with 10% goat serum and incubated overnight at 4°C with rabbit anti-REDD1 antibody (1:100 dilution; Proteintech). After washing with phosphate buffered saline (PBS), sections were incubated for 30 minutes with ImmPRESS-AP anti-rabbit IgG polymer detection reagent (Vector Laboratories), dehydrated and mounted. Positive cells were counted in 2 different fields per section and the results are reported as percentage of REDD1-positive cells.

Paraffin-embedded knees from REDD1^+/+ mice were processed as described (9 (link)) and sections were incubated overnight with antibodies against REDD1 (1:100, Proteintech), LC3 (1:50, MBL) or ATG5 (1:100, ABCAM). After washing with phosphate buffered saline (PBS), sections were incubated for 30 minutes with ImmPRESS-AP anti-rabbit IgG polymer detection reagent (Vector Laboratories), dehydrated and mounted. Positive cells were quantified in the articular cartilage and both meniscal horns of the proximal tibial plateau in two different pictures per section taken at 20x magnification. Results are reported as percentage of immunopositive cells.