Cd3 clone m 20

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.