Anti cleaved caspase 3 asp175 9 661

Renal cortical and outer medulla tissues were lysed using 2% SDS buffer with 1% protease inhibitor cocktail (P8340, Sigma-Aldrich). Protein concentration was determined using a Pierce BCA protein assay kit (no. 23225) from Thermo Scientific. Equal amounts of proteins were separated by SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% fat-free milk or 5% BSA for 1 h and subsequently incubated with primary antibodies at 4°C overnight and secondary antibodies for 1 h at room temperature. Primary antibodies used in present study were from following sources: anti-Vimentin 5,741) and anti-cleaved Caspase-3 (Asp175) 9,661) from Cell Signaling Technology; anti-LC3B (NB100-2220) from Novus Biologicals; anti-α-Smooth Muscle Actin (ab5694) and anti-Fibronectin (ab2413) from Abcam; anti-GAPDH (10494-1-AP) from Proteintech; anti-Collagen 1 (AF7001) from Affinity; all secondary antibodies for immunoblot analysis from Thermo Fisher Scientific. Antigen-antibody complexes on the membranes were detected with an enhanced chemiluminescence kit from Thermo Scientific. For quantification, protein bands were analyzed with ImageJ software.

Hematoxylin and eosin (H&E) and IHC stainings were performed at the Mouse Pathology Facility of Lausanne University (Epalinges, Switzerland). IHC labeling was performed using the mouse monoclonal anti-human vimentin Ab (clone RV202, GeneTex, CA, USA), anti-Ki67 (clone MIB-1, Dako, Denmark), and anti-cleaved caspase-3 (ASP175)(#9661, Cell Signaling Technology Inc.) on 4 μm-thick paraffin-embedded mouse tissue sections.

Dissected intestinal tissues were fixed with 4% paraformaldehyde, embedded in paraffin wax and sectioned at a thickness of 4 µm. These sections were stained with H&E or were processed for further immunostaining as described previously (Oshima et al., 1995b (link)). Rabbit polyclonal anti-cleaved caspase3 (Asp175, #9661; Cell Signaling Technology), anti-Ki67 (RM-9106; Thermo Fisher Scientifics) were used as primary antibodies.