Goat anti rat igg h l alexafluor 488

Mouse kidneys were collected at 28 days after ADR administration. Renal tissues were frozen with liquid nitrogen in OCT compound immediately after collection. Frozen tissues were sliced into 10-μm thickness using cryostat and attached onto glass slides. Sections were thawed with ice-cold acetone at − 20 °C for 5 min. After blocking with Protein Block Serum-Free (Dako, USA) at room temperature for 30 min, sections were reacted with primary antibody diluted at 1:100 and secondary antibody diluted at 1:300 with Antibody Diluent with Background Reducing Components (Dako, USA). Primary antibody was reacted overnight at 4 °C and secondary antibody was reacted for 1 h at room temperature. Polyclonal rabbit anti-WT-1 (sc-192; Santa Cruz Biotechnology), purified rat anti-mouse CD44 (#550538; BD, USA), Goat anti-rabbit IgG (H + L) Alexa Fluor 488 (A-11008; Thermo Scientific Inc., USA) and Goat anti-rat IgG (H + L) Alexa Fluor 488 (A-11006; Thermo scientific Inc., USA) were used for antibody reactions. Sections were incubated with 1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) solution for 20 min at room temperature. Then, stained sections were mounted with VECTASHIELD mounting medium (Vector Laboratories, USA). Images were captured using BIOREVO microscope (Keyence, Japan).

Frozen sections 6–8 μm thick were fixed in ice‐cold acetone, rehydrated in PBS and blocked with normal horse serum before antibody application. The following antibodies were purchased from BioLegend: anti‐CD1d (1B1), anti‐CD4 (RM4‐5), anti‐CD21/35 (7E9) and anti‐CD45R/B220 (RA3‐6B2). The following antibodies were purchased from BD Biosciences: anti‐CD35 (8C12), anti‐MAdCAM‐1 (MECA‐367) and anti‐TNP (G235‐1). Anti‐CD169 (MOMA‐1) and anti‐MARCO (ED31) were purchased from Bio‐Rad (Hemel Hempstead, UK). Anti‐CD209b/SIGNR1 (eBio22D1) and phycoerythrin (PE)‐conjugated anti‐Armenian hamster IgG were purchased from eBiosciences (ThermoFischer, Loughborough, UK). Anti‐CXCL13 (polyclonal) was purchased from R&D Systems. Streptavidin Alexa Fluor 594, goat anti‐rat IgG (H+L) Alexa Fluor 594, donkey anti‐goat IgG (H+L) Alexa Fluor 647 and goat anti‐rat IgG (H+L) Alexa Fluor 488 were purchased from ThermoFisher Scientific (Waltham, MA). Dako fluorescent mounting medium (Agilent, Santa Clara, CA) was used to apply coverslips before image acquisition. A Zeiss LSM5 Pascal (Carl Zeiss, Oberkochen, Germany) upright microscope with zen software (Rochdale, UK) was used for image collection.