Zymosan a

Fluorescein sodium salt was purchased from Sigma-Aldrich. Dextran (Dex)-FITC (70 kDa), LPS-AF488, fluorescent latex beads (FluoSphares, 0.1 µm, yellow-green), and S. aureus-AF594 were purchased from Thermo Fisher Scientific. OVA (chicken, Sigma) and zymosan A (Wako) were labeled with FITC (Pierce, 46,110). Briefly, OVA (10 mg/ml) or zymosan (20 mg/ml) was dissolved in 0.5 mg/ml FITC, 10 mM Na₂CO₃, 100 mM NaCl (pH8.0) and incubated at 37°C or room temperature for 1 h. The FITC-labeled OVA was dialyzed in PBS. FITC-labeled zymosan was washed five times with PBS by centrifugation and stored at −20°C until use.
Fluorescent tracers were subcutaneously injected into the fore footpads, hind footpads, shoulders, chest, flanks, lower abdomens, and tail bases, or alternatively only to the left hind footpad, or left tail base for analyzing individual LNs in a lymphatic basin from single-site drainage. LNs were excised 1 h after injection of fluorescent tracers.

6-wk-old BALB/c mice were immunized subcutaneously (s.c.) with His-tagged CBX3 protein (200 μg) emulsified with CFA (500 μg) or CFA alone, and once again 3 wk later. 4-wk-old BALB/c mice were immunized s.c. with UV-killed C42 or E. faecalis (1.5 × 10¹⁰) and zymosan (4 mg/ml; Zymosan A; Wako) as an adjuvant (Nihei et al, 2023b (link)). 4 wk after the final immunization, sera were collected from mice and used for WB analysis.