Anti 4 hydroxynonenal antibody ab46545

IF was performed on paraffin embedded sections (5 µm) as previously described 23 . Briefly, sections were deparaffinized in xylene and ethanol before antigen retrieval in sodium citrate buffer (10 mM, pH 7.5) for 20 min at 90 ° C. Cultured cells were plated on coverslips, cultured overnight and treated the next day. At collection cells were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100/PBS. Acrolein antibody (Invitrogen MA527553 Waltham, MA) was used at 1:50 and anti-4-Hydroxynonenal antibody (ab46545 from Abcam Cambridge, MA) was used at 1:100 and incubated overnight at 4 ° C, anti-γH2A.X (Cell Signalling Rabbit mAb#9718) was used at 1:500. Secondary goat anti-rabbit Daylight 488 (Fisher PI35552) was used at 1:500 and incubated for 45 min in the dark at room temperature. Following 3 washes, slides were then mounted with DAPI mounting media. Images were captured using the Leica DM4 B upright microscope (Leica Chicago, IL).