Biotinylated rabbit anti rat

Paraffin sections of formalin fixed kidneys were cut (1~2 μm) and incubated with anti-B220 (clone RA3-6B2, eBioscience Hatfield UK) or subjected to a heat-induced epitope retrieval step prior to incubation with primary antibody for CD3 (clone M-20, Santa Cruz Biotechnology, Dallas, Texas, USA), CD4 (clone 4SM95, eBioscience Hatfield UK) or MPO (polyclonal rabbit, Dako Denmark, Glostrup Denmark) followed by incubation with biotinylated rabbit anti-rat, rabbit anti-goat or donkey anti-rabbit (Dianova, Hamburg, Germany). For detection, sections were incubated with alkaline phosphatase (AP) labelled streptavidin (Dako Denmark, Glostrup Denmark). AP was visualized using Fast Red as chromogen (Dako Denmark, Glostrup Denmark). For detection of regulatory T cells, sections stained for CD4 were subjected to protein inactivation step prior to incubation with anti-Foxp3 (clone FJK-16s, eBioscience Hatfield UK). For detection, EnVision+ System- HRP Labelled Polymer Anti-Rabbit (Dako Denmark, Glostrup Denmark) was used. HRP was visualized with diaminobenzidine (Dako Denmark, Glostrup Denmark) as chromogen. Nuclei were counterstained with hematoxylin and slides coverslipped with glycerol gelatine (Merck, Kenilworth, NJ, USA). Negative controls were performed by omitting the primary antibody.

Immunopathological analysis was provided by the iPATH.Berlin—Immunopathology for Experimental Models, core unit of the Charité—Universitätsmedizin Berlin (Berlin, Germany). Formalin-fixed tissue samples were embedded in paraffin and 1–2 μm thick sections were cut from paraffin blocks. Sections were dewaxed and histochemically stained with H&E (hematoxylin&eosin) for evaluation of histomorphology or subjected to a heat-induced epitope retrieval step prior to incubation with anti-Ki67 (clone 16A8, BioLegend), anticleaved caspase 3 (clone 5A1E, Cell Signaling). For detection of Ki67 and cleaved caspase3, sections were incubated with secondary antibody (biotinylated rabbit antirat and biotinylated donkey antirabbit, respectively; Dianova). Biotin was detected by streptavidin coupled with alkaline phosphatase (AP) and RED as chromogen (both Dako REAL™ Detection System, Alkaline Phosphatase/RED, Rabbit/Mouse, Agilent Technologies). Nuclei were counterstained with hematoxylin (Merck Millipore) and slides coverslipped with glycerol gelatine (Merck). Whole slide scans were acquired by a Vectra® 3 imaging system (PerkinElmer).