Vectastain elite abc standard kit

At 3 or 7 days after MCAO, the brains were perfused and fixed overnight with 4% paraformaldehyde. Fixed brains were sliced into 20 µm thick sections using a cryostat (CM3050S, Leica). The brain sections were treated with blocking solution (10% horse serum in PBS) for 1 h at 20–25 °C, incubated overnight at 4 °C with the biotinylated anti-mouse IgG (H + L) (1:100; BA-2000, Vector Laboratories) in 10% horse serum in PBST. The VECTASTAIN Elite ABC Standard Kit (PK-6100, Vector Laboratories) was used as a signal enhancer, and DAB colour development was performed. Images were taken, combined, and analysed using a digital microscope (BZ-X800 Viewer/Analyzer 1.1.2.4, KEYENCE). IgG staining was quantified by integrating the intensity values of the 12 sections from each mouse.

To characterize the cellular morphology of t. palatina in each species, immunohistochemistry was applied using the avidin-biotin-peroxidase-complex (ABC) method utilizing the Vectastain® Elite ABC standard kit (Vector Laboratories) with citric buffer (10 mM, pH 6,0) pre-treatment, to label the following tonsillar epitopes: cytokeratins 1–8, 10, 13–17, 19 (clone AE1/AE3, mouse anti-human, monoclonal, diluted 1:500, Dako, Deutschland GmbH, Hamburg, Germany), macrophages and dendritic cells (anti IBA1, rabbit anti-human, polyclonal, diluted 1:200, FUJIFILM Wako Chemicals, Germany), B cells (CD20, rabbit anti-human, polyclonal, diluted 1:200, Thermo Fisher, Germany), T cells (CD3, rabbit anti-human, polyclonal, diluted 1:200, Dako, Germany,) and RABV-nucleoprotein (polyclonal, rabbit-α-N161-5; diluted 1: 2000^{73 (link)}) by incubating overnight. Antigen visualization was performed with 3-amino-9-ethyl-carbazol as chromogen and hematoxylin as counterstain. As negative controls, consecutive sections were incubated with rabbit serum or tris-buffered saline instead of the primary antibodies.

Ki67 immunostaining was performed on paraffin-embedded sections treated with HistoVT One (Nacalai Tesque, Kyoto, Japan) at 105 °C for 15 min for antigen retrieval. According to the manufacturer’s instructions, tissue sections were incubated with anti-Ki67 primary antibodies (RM-9106; LabVision, Fremont, CA, USA) overnight at 4 °C. Bound primary antibodies were detected using biotinylated goat anti-rabbit IgG antibodies (Vector Laboratories, Burlingame, CA, USA) and the Vectastain Elite ABC standard kit (Vector Laboratories). Tissue sections were stained using DAB-H₂O₂ as a substrate.
To directly measure DNA synthesis, the EdU assay was performed. EdU solution was prepared by dissolving 50 mg of EdU (Invitrogen, Carlsbad, CA, USA) in 50 mL of PBS. Mice were administered intraperitoneal injections of EdU (10 mg/kg body weight) 8 h before kidney harvest. The EdU assay was performed using Click-iT^® EdU Imaging Kits (Invitrogen), according to the manufacturer’s recommendations. EdU-positive cells were counted separately in 10 randomly selected non-overlapping renal corticomEdUllary fields (400× magnification) of tubular or interstitial areas per section in each mouse. The results were expressed as the number of EdU-positive cells per field in the tubular region and in the interstitial region.

For immunohistochemical staining, 4 μm paraffin-embedded sections were deparaffinized with xylene and washed in ethanol. The slides were incubated with 0.3% H₂O₂ solution (diluted in distilled water) for 10 minutes to quench endogenous peroxidase activity. After rinsing in phosphate‑buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄ solution, pH 7.6), the sections were blocked in 1× Block Ace solution (Dainippon Pharmaceutical, Osaka, Japan) at room temperature for 1 hour and incubated overnight at 4°C in the partially purified rabbit anti–ITPA-N antibody (0.5 μg/mL in PBS). After rinsing in PBS, the sections were incubated with a biotinylated goat anti–rabbit IgG antibody (VECTOR Laboratories, Burlingame, California, USA) at room temperature for 45 minutes. VECTASTAIN Elite ABC Standard Kit (VECTOR Laboratories) and DAB Substrate Kit (VECTOR Laboratories) were then used to visualize the bound secondary antibody. Digital images were acquired using an Axio Imager A1 microscope, equipped with an AxioCam charge-coupled device camera and the AxioVision 4.9 imaging software program (Carl Zeiss Microscopy, Tokyo, Japan). Views of entire coronal sections were obtained using a Nikon Eclipse 80i microscope with a Virtual slice module in the Stereo Investigator software program (MBF Bioscience, Williston, Vermont, USA).