Goat anti mouse igm af568

Goat anti-mouse IgG (H+L) AF488 (ThermoFisher, 1:400), goat anti-rabbit IgG (H+L) AF647 (ThermoFisher, 1:400), goat anti-mouse IgM AF568 (ThermoFisher, 1:400) and IRDye 800CW goat anti-rabbit IgG (LI-COR, 1:5,000)

Rats were anesthetized with an overdose of pentobarbital and perfused with PBS and 4% paraformaldehyde (PFA). Spinal cords were dissected and post-fixed in 4% PFA overnight at 4 °C, rinsed with PBS and dehydrated in 20% sucrose. 20 μm-thick sections were mounted on SuperFrost slides after cryosectioning. For iNOS staining, heat mediated antigen retrieval was performed prior to blocking by incubating the slides in citrate buffer pH 6.0 at 90 °C for 30 min. After blocking, sections were incubated with primary antibodies overnight at 4 °C and followed by appropriate secondary antibodies. Primary antibodies used are detailed in Supplementary Table 4. The sections were incubated with appropriate fluorescently conjugated secondary antibodies: goat anti-mouse IgM AF-568 (#A21043, Thermo, 1: 500), donkey anti-rabbit IgG AF-568 (#A10042, Thermo, 1:500) and coversliped using Fluoromount-G with DAPI mounting medium. For each staining, four individual animals per group were examined and images were captured with a ZEISS Imager Z1 fluorescence microscope equipped with a AxioCam MRm camera. Tile scans were stitched in post-acquisition processing using AxioVision software. Staining was quantified using ZEISS ZEN software by measuring the mean fluorescence over sections excluding roots and meninges.