CXCR5 Immunohistochemistry in Tissues

Eyeballs were fixed with HistoChoice Molecular Biology fixative (H120–4L, VWR Life Science, Radnor, PA, USA) for 12 h and stored in PBS (10010023, Thermo Fisher Scientific) until the specimens were processed for paraffin embedding and sectioning (5-μm thick). The sections were rehydrated using gradations of xylene and ethanol. Heat-induced antigen retrieval was performed in a citrate buffer (pH: 6.0), and sections were depigmented in 10% H₂O₂ for 120 min at 65 °C [21 (link)]. The sections were then blocked with 5% bovine serum albumin (BSA; A7096) at RT for 1 h and then incubated with primary CXCR5 antibodies (Table 1) overnight at 4 °C. Following PBS-Tween 20 (0.05%; PBS-T) washing, horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000; Biorad) was applied for 1 h. The CXCR5 signals were visualized by incubation with a working solution of the DAB substrate kit (34002; Thermo Fisher) for 7 min. The reaction was stopped by briefly washing the samples with PBS.