Rodent block m rbm961g

Formalin-fixed, 3-μm paraffin-embedded kidney sections were incubated with Peroxidazed 1 (PX968H, Biocare Medical, Pacheco, CA, USA), after antigen retrieval in a decloaking chamber with Rodent decloaker (RD913M, Biocare Medical) buffer. After blocking for 30 min with Rodent Block M (RBM961G, Biocare Medical), sections were incubated with rabbit anti- Wilms Tumor 1 (WT1, 1:600; ab89901, abcam, Cambridge, UK) antibody followed by Rabbit on Rodent horseradish peroxidase (HRP)-Polymer (RMR622G, Biocare Medical,) for 30 min at room temperature. Staining was visualized using diaminobenzidine (BDB2004H, Biocare Medical) substrate solutions. Slides were counterstained with Mayer’s hematoxylin (MHS80-2.5L, Bio Optica, Milan, Italy), mounted with Eukitt mounting medium (09-00250, Bio Optica) and finally observed using light microscopy (ApoTome, Axio Imager Z2, Zeiss, Oberkochen, Germany). Negative controls were obtained by omitting the primary antibody on adjacent sections. At least 15 glomeruli/section for each animal were randomly acquired. The average number of podocytes per glomerulus and the glomerular volume were estimated using morphometric analysis, as previously described [28 (link)].

Segments of distal ileum (1 cm of intestine withdrawn above the junction with the caecum) were harvested from all rats and fixed in 10% neutral-buffered formalin for 24 h, dehydrated and embedded in paraffin. The paraffin-embedded tissue sections were cut (3 μm thickness) and mounted on adhesive microscope slides. The sections were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol (100%, 95% and 70%) and distilled water. Antigen retrieval was performed in 0.01 M sodium citrate buffer with 0.05% Tween 20 (pH 6.0), using a water bath at 98 °C for 10 min and blocked in 3% H₂O₂ in PBS. After that, sections were incubated with a blocking solution (Rodent Block M, RBM961G, Biocare Medical, UK) for 30 minutes and then incubated overnight at 4 °C with anti-GLP-1 primary antibody: mouse monoclonal, ab26278; Abcam, Amsterdam, The Netherlands; 1:1500. Subsequently, sections were incubated with secondary antibody at RT for 30 min: goat anti-mouse, ab97021; Abcam, Amsterdam, The Netherlands; 1:200. Then, 30-minute ABC complex incubation was carried out (Vector A and B, Vectastain, Vector Labs, Peterborough, UK). Finally, the sections were treated with diaminobenzidine tetrahydrochloride (DAB) substrate kit (ab64238, Abcam, Amsterdam, The Netherlands). All sections were stained with H&E.