MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2

McCarthy, Mary K.; Reynoso, Glennys V.; Winkler, Emma S.; Mack, Matthias; Diamond, Michael S.; Hickman, Heather D.; Morrison, Thomas E.

Lymph nodes were fixed in 1 mL of phosphate buffer containing 0.1 M L-lysine, 2% PFA, and 2.1 mg/mL NaIO 4 at pH 7.4 for 24 h at 4˚C, followed by incubation in 30% sucrose phosphatebuffered solution for 48 h, then in 30% sucrose/PBS for 24 hr. LNs were then embedded in optimal-cutting-temperature medium (Electron Microscopy Sciences) and frozen in dry-icecooled isopentane. Eighteen-μm sections were cut on a Leica cryostat (Leica Microsystems). Sections were blocked with 5% goat, donkey, bovine, rat or rabbit serum and then stained with one or more of the following: B220 (clone RA3-6B2, ThermoFisher), CD8α (clone 53-6.7, ThermoFisher), ERTR-7 (Rat monoclonal, BioXCell), Gr-1 (clone RB6-8C5, BioXCell), and GL7 (clone GL7, BioLegend). Images were acquired using identical photomultiplier tube (PMT) and laser power settings on a Leica SP5 confocal equipped with HyD detectors (Leica). Confocal microscopy images were performed of the entire popliteal lymph node (representing approximately a 7 mm 2 imaged area) and individual fields (tiles) were merged into a single image file. Images were analysed using Imaris v9.02 software (Bitplane). The total number of GCs, area per GC, and total GC area were calculated per 18-μM dLN section automatically using Imaris' surfaces function based upon GL7 staining. The total GC number was also confirmed manually.