Following sacrificing by CO2 asphyxiation, one half of a kidney was used for histological assessment of classical markers of glomerular damage and morphometric changes to confirm diabetic nephropathy. For this, capillary and mesangial matrix area were quantified in Periodic acid-Schiff (PAS) or Trichrome-stained paraffin embedded 4 μm sections, respectively. We used a semiautomatic image analyzing system (Leica Q600 Qwin; Leica Microsystems, Cambridge, UK) to determine the fraction of glomerular surface area by the point-counting method.
Glomerular heparanase expression was quantified after overnight incubation with primary antibody (Polyclonal rabbit anti-heparanase 1.5 μg/mL, InSight Biopharmaceuticals, Rehovot, Israel), followed by goat anti-rabbit IgG-Alexa 594 (1/1000), for 1 hour, both in blocking buffer. Sections were counterstained with Hoechst (1/1000) and embedded in Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA). Cathepsin L polyclonal antibody (R&D Systems) was incubated overnight, followed by horseradish peroxidase–conjugated secondary antibody and 3,3’-diaminobenzidine and counterstained with hematoxylin. Staining area was quantified as the percentage of stained area divided by the glomerular area.
Glomerular heparanase expression was quantified after overnight incubation with primary antibody (Polyclonal rabbit anti-heparanase 1.5 μg/mL, InSight Biopharmaceuticals, Rehovot, Israel), followed by goat anti-rabbit IgG-Alexa 594 (1/1000), for 1 hour, both in blocking buffer. Sections were counterstained with Hoechst (1/1000) and embedded in Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA). Cathepsin L polyclonal antibody (R&D Systems) was incubated overnight, followed by horseradish peroxidase–conjugated secondary antibody and 3,3’-diaminobenzidine and counterstained with hematoxylin. Staining area was quantified as the percentage of stained area divided by the glomerular area.