A21125

Methods used for serologic assays to assess antibody responses have been described in detail previously (24 (link)). Plasma was isolated within 12 hours of collection prior to serologic testing with the EUROIMMUN or Abbott ARCHITECT assays. The EUROIMMUN anti–SARS-CoV-2 IgG ELISA was performed according to the manufacturer’s protocol. The cutoff for positivity was an OD ratio ≥ 1.1 based on the manufacturer-provided reference. The Abbott SARS-CoV-2 IgG chemiluminescent microparticle immunoassay (Abbott ARCHITECT) was performed according to the manufacturer’s instructions. A kit calibrator was used to generate the assay index, with values ≥ 1.4 considered positive. nAb titers were measured in plasma that was frozen within a few hours of specimen collection, shipped on dry ice, and then thawed at the time of the assay. nAb titers were assessed using a fluorescence reduction neutralization assay assessing inhibition of SARS-CoV-2 replication in Vero E6 cells (CRL-1586, ATCC). SARS-CoV-2 (2019-nCoV/USA-WA1-A12/2020, US CDC) was detected using a primary antibody targeting nucleoprotein (40143-MM05, Sino Biological) and an Alexa Fluor 594–conjugated secondary antibody (A-21125, Invitrogen). Plasma dilutions of 1:40 that did not result in at least a 50% reduction in viral titer are reported as undetectable (<1:40), as previously described (53 (link)).

Sections were blocked in 2% bovine serum albumin (BSA) plus M.o.M. (MKB‐2213, Vector Labs) for 60 min. Sections were then incubated in primary antibodies against eMyHC (supernatant 1:20; F1.652, Developmental Studies Hybridoma Bank) and laminin (1:200; L9393, Sigma‐Aldrich) diluted in 2% BSA for 90 min, washed in PBS, and incubated in secondary antibodies against Ms IgG1 AF594 (1:200; A‐21125, Invitrogen) and Rb IgG AF488 (1:200; A‐11008, Invitrogen) diluted in PBS for 60 min. Sections were washed, incubated in DAPI (1:10,000; D1306, Invitrogen) for 15 min, washed in PBS, and cover slipped using PBS and glycerol (1:1).