Avidin biotin complex peroxidase standard staining kit

Renin protein expression was assessed by immunohistochemistry. Paraffin blocks were cut at 4 µm, deparaffinized in xylene, and rehydrated through graded alcohols. After deparaffinization, endogenous peroxidases were blocked in an aqueous solution containing 3% H₂O₂ and 10% methanol. Antigen retrieval was performed boiling the samples in citrate buffer (10 mM citric acid pH 6.0). The sections were then blocked in bovine serum albumin (5%) and incubated during 16 h at 4 °C with anti-renin primary antibodies (Dilution 1:2000, ab212197, Abcam, Cambridge, UK). Biotinylated antibodies against rabbit IgGs (Dilution 1:250, BA-9500, Vector Laboratories, Burlingame, CA, USA) were employed as secondary antibodies. Proteins were visualized using the Avidin-Biotin Complex (ABC) Peroxidase Standard Staining Kit (32020, ThermoFisher Scientific, Waltham, MA, USA) followed by 3,3′-Diaminobenzidine (DAB) Enhanced Liquid Substrate System (D3939, MilliporeSigma, Darmstadt, Germany). Counterstaining was done with Haematoxylin Gill Nº3 solution. Once stained, 10 representative images of the juxtameglomerular apparatus per section were taken at 400× magnification to measure renin stained area.

The brains of mice were removed and fixed in phosphate-buffered 4% paraformaldehyde (pH 7.4). Then, the fixed samples were sectioned in the coronal plane at 40 μm thickness using a cryomicrotome (Leica, Wetzlar, Germany). The obtained sections were incubated with 0.01 M sodium citrate buffer (pH 6.0) for 30 min at 90°C and then washed with Tris-buffered saline with Tween (TBST). Next, the sections were blocked in 10% goat serum for 1 h and then incubated with anti-BrdU (1:1,000), anti-DCX (1: 500), anti-Sox-2 (1:200), or anti-p-CREB (1:1,000) overnight at 4°C. Staining was revealed using biotinylated secondary antibodies and avidin-biotin complex (ABC) peroxidase standard staining kit (Thermo Fisher Scientific) with diaminobenzidine (DAB, Thermo Fisher Scientific). Immunopositive cells were quantified using Fiji ImageJ software (https://fiji.sc/). The total number of BrdU, DCX, and Sox-2-immunopositive cells (BrdU⁺, DCX⁺, and Sox-2⁺) per DG was estimated based on the method described previously (17 (link)), while p-CREB immunoreactivity in the granule cell layer (GCL) of the DG was quantified by using the IHC Profiler plugin for ImageJ according to the procedures reported by Varghese et al. (18 (link)). For each marker, 5 sections of the DG from an individual animal were randomly selected and analyzed by the same observer, who was unware of animal assignments.

Wilms Tumor 1 (WT1) was detected by immunohistochemistry to count the number of podocytes per glomerular area. Paraffin blocks were cut at 4 µm, deparaffinized in xylene and rehydrated through graded alcohols. After deparaffinization, endogenous peroxidases were blocked in an aqueous solution containing 3% H₂O₂ and 10% methanol. Antigen retrieval was performed boiling the samples in citrate buffer (10 mM citric acid pH 6.0). The sections were then blocked in bovine serum albumin (5%), and incubated during 16 h at 4 °C with anti-WT1 primary antibodies (Dilution 1:100, ab89901, Abcam, Cambridge, UK). Biotinylated antibodies against rabbit IgGs (Dilution 1:250, BA-9500, Vector Laboratories, Burlingame, CA, USA) were employed as secondary antibodies. Proteins were visualized using the Avidin-Biotin Complex (ABC) Peroxidase Standard Staining Kit (32020, ThermoFisher Scientific, Waltham, MA, USA) followed by 3,3′-Diaminobenzidine (DAB) Enhanced Liquid Substrate System (D3939, MilliporeSigma, Darmstadt, Germany). Counterstaining was done with Haematoxylin Gill Nº3 solution. Podocyte density was evaluated in 20 representative cortical glomeruli and expressed as number of podocytes per mm² of glomerular area.