Rabbit anti rat biotinylated

Spleen and livers were fixed overnight in 4% formalin in PBS and embedded in paraffin using routine procedures. Then, 5 µm sections were deparaffinised with xylene, incubated in 3% H₂O₂ in methanol for 20 min to block endogenous peroxidase, and subsequently rehydrated using a descending ethanol series. Sections were incubated in 10 mM citrate buffer (pH 6.0) at 37 °C for 2 h and subsequently incubated in 0.075% trypsin (Sigma-Aldrich) in PBS at 37 °C for 7 min. Nonspecific binding was blocked by incubating the sections in 10% normal rabbit serum in PBS for 20 min and subsequently the sections were incubated overnight at 4 °C with an anti F4/80 antibody 1:1000 in PBS, 2% NRbS (Rat anti ms, hu F4/80;BM8 eBioscience). After washing with PBS, the sections were incubated for 30 min at RT with 200 times diluted biotinylated rabbit-anti-rat (Vector laboratories, cat.nr. BA-4001). Finally, an avidin–biotin complex was applied to the sections for 30 min, and they were subsequently incubated with Bright DAB (3,3′-diaminobenzidine) for 8 min at RT. Nuclei were stained by incubating the sections for 5 s with hematoxylin and a subsequent wash with tap water for 10 min. Finally, the sections were dehydrated with, consecutively, water, 50%, 70%, and twice 100% ethanol, and twice with xylene, after which they were mounted with Permount, dried, and imaged.