Avidin biotin peroxidase complex

Free-floating DMPFC brain sections were immunolabeled for NECAB2 (Thermo Fisher Scientific, Cat. No. PA5-53108). The antibody (1:250 dilution) was applied for 24 h at room temperature, followed by incubation of the sections in biotinylated anti-rabbit secondary antibody (1:1000 dilution, Vector Laboratories, Burlingame, CA, USA) and then in avidin–biotin–peroxidase complex (1:500, Vector Laboratories) for 2 h. Subsequently, the labeling was visualized via incubation in 0.02% 3,3-diaminobenzidine (DAB; Sigma), 0.08% nickel (II) sulfate and 0.001% hydrogen peroxide in PBS, pH 7.4 for 5 min. Sections were mounted, dehydrated and coverslipped with Cytoseal 60 (Stephens Scientific, Riverdale, NJ, USA).

Histological sections of renal tissue were incubated for 1 hour at room temperature with monoclonal anti-ED1 antibody (1 : 300) and overnight at 4°C with the antibodies anti-CD43 monoclonal (1 : 200) and anti-NF-κB p65 polyclonal (1 : 100) [13 (link)]. The reaction product was detected with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, USA), and the color reaction was developed with 3,3-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). The sections were then counterstained with methyl green or hematoxylin. Negative controls consisted of replacing the primary antibodies with equivalent concentrations of normal rabbit IgG. Except for ED-1, nonspecific protein binding was blocked by incubation with 20% goat serum in PBS for 30 min. We evaluated the immunostaining for NF-κB (p65) using ImageJ software, and the results were expressed as percentages, while the quantification for ED-1 and CD43 was performed by counting positive cells in 30 fields (0.245 mm²) of the tubulointerstitial compartment of each histological slide.

Pancreases were paraffin embedded and prepared for histological analysis (Wang and Brubaker, 2002 (link)). Briefly, freshly isolated pancreas was cut into 8–10 segments followed by formaldehyde fixation. All pancreatic pieces were embedded in paraffin after being dehydrated in ethanol and cleaned with xylene. Insulin and glucagon dual staining were performed on tissue sections (5 μm) by using guinea pig anti-insulin and rabbit anti-glucagon antibodies (1:1000; DAKO, Burlington, ON, Canada); then detected with fluorescent (Cy3- and FITC- conjugated IgG) or biotinylated secondary antibodies (1:200; Abcam, Cambridge, United Kingdom). For IHC staining, samples were incubated with avidin-biotin-peroxidase complex (Vector Laboratories, Burlington, ON, Canada) before staining with DAB (Vector Laboratories) or Fuchsin red (DAKO) and subsequent hematoxylin counterstaining. Entire pancreatic images were scanned and viewed with NanoZoomer (Hamamatsu, Hamamatsu-shi, Shizuoka-ken, Japan) and analyzed by using the ImageScope program (Aperio Technologies, Vista, CA, United States) (Purwana et al., 2014 (link)). Total alpha and beta cell mass were determined by the product of cross-sectional alpha and beta cell area over total tissue area and the weight of pancreatic tissue before fixation.