3 3 diaminobenzidine dab substrate

Kidneys were weighed and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). The fixed kidneys were rinsed with PBS, dehydrated, embedded in paraffin, and sectioned into 2–4 μm thick sections for hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Masson’s trichrome (MT) staining [17 (link)]. Renal tubule lumen areas were measured using Image J software. The kidney sections were rehydrated, autoclaved at 105 °C for 5 min, blocked by incubation with 10% BSA in PBS for 30 min, and incubated with 0.3% hydrogen peroxide in PBS to inactivate any endogenous peroxidases. The sections were subsequently incubated with mouse anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 °C, rinsed with PBS, and incubated with horseradish peroxidase (HRP)-conjugated donkey anti-mouse antibody (1:1000, Santa Cruz Biotechnology Inc.) for one hour at room temperature. Immunoreactivity was visualized with 3,3′-diaminobenzidine (DAB) substrate (BD Pharmingen, San Diego, CA, USA). The number of PCNA-positive cells was estimated as an average of counts in 10 pictures.

Pancreas sections were rehydrated, blocked with 3% BSA in PBS for 30 min, then incubated with 0.3% H₂O₂ in PBS to inactivate any endogenous peroxidase activity. Sections were next incubated with guinea pig anti-insulin polyclonal antibody (1 : 100) overnight at 4°C, rinsed with PBS, and incubated with horseradish peroxidase- (HRP-) conjugated goat anti-guinea pig antibody (1 : 400, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for one hour at room temperature. Immunoreactivity was visualized with 3,3′-diaminobenzidine (DAB) substrate (BD Pharmingen, San Diego, CA), and insulin-positive areas were measured using ImageJ software.