Ab15580

For IHC, paraffin-embedded sections were stained with antibodies against p62 (Enzo Life Sciences, PW9860-0100), Ki67 (Abcam, ab-15580), pS6 (Cell Signaling, 4858S), p42/44 MAPK (p-ERK) (Cell Signaling, 4376), and 4-hydroxynonenal (4-HNE) (Abcam, ab46545) as described previously [30 (link)].
For quantification of IHC for Ki67, pS6, p-ERK and 4-HNE, 6–10 representative images from each treatment group were obtained and scored using the ImageJ software.

Cells cultured in vitro were fixed in 4% paraformaldehyde at room temperature for 25 min or heart tissues were dissected and fixed in 4% paraformaldehyde at 4°C overnight. The fixed heart tissues were washed three times with PBS and equilibrated in 30% sucrose at 4°C for 2 days before freezing and cryosectioning. Eight micrometer frozen sections were prepared. Cells or tissue sections were blocked with 5% BSA and 5% goat serum and then stained with the respective primary antibodies at 10 ug/ml at 4˚C overnight. Anti-mouse primary antibodies used: COLA1 (abcam, ab34710), cTnT (abcam, ab8295), pH3 (Millipore, 06-570), Ki67 (abcam, ab15580) or cCASP3 (Cell signaling technology, 9661). Alexa-Fluor-488- or Alexa-Fluor-546-conjugated secondary antibodies (Invitrogen) were used at room temperature for 30 min in the dark. For cell death analysis, an in situ cell death detection kit by TUNEL was also used per manufacturer's instruction (Roche, 1256792910). Slides were mounted with DAPI-containing fluorescence mounting medium (Dako) and fluorescence was detected with an upright fluorescence microscope, inverted fluorescence microscope or confocal microscope (all Leica). Images were processed with the ImageJ software and cTNT coverage was analyzed based on this formula: cTNT⁺ area/total area.