Histochoice molecular biology fixative

The eyeballs were fixed with HistoChoice Molecular Biology fixative (VWR Life Science) for 12 hours at 4C and stored in PBS until being processed for paraffin embedding and sectioning (5 μm thick). As the GFP fluorescence was reduced significantly by tissue fixation and processing, sections were rehydrated, and the GFP signal was enhanced by incubation overnight at 4 °C with GFP antibody (Table 1), following by incubation with Goat anti-Rabbit IgG (H+L), Cyanine5 (A10523, 1:1000; Thermo Fisher Scientific, Waltham, MA, USA), secondary antibody for 1 h at RT, and counterstained with DAPI 1:5000 (Sigma-Aldrich, St Louis, MO, USA) and mounted with the ProLong Diamond antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA). Sections were imaged using the EVOS FL fluorescent microscope (Thermo Fisher Scientific, Waltham, MA, USA).

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.