Fix perm intracellular staining buffers

Spinal cords at the sacral level from naïve and EAP mice were homogenized and passed through a 40μm Cell strainer (Fisher Scientific, Cat No. 22363547) to generate single-cell suspensions. Staining for resident microglia (CD11b, CD39), monocytes (CD11b, Ly6C), and CCL2. For labeling surface marker, fluorescent dyes-conjugated antibodies were incubated for 30 min in the dark at 4°C. For labeling intracellular marker, cells were permeabilized prior to intracellular staining. Intracellular staining was performed by fixation and permeabilization using eBioscience Fix-Perm Intracellular staining buffers (Cat. Num 8222-49 and 8333-56). Staining was performed for 1 hour at room temperature, followed by washing in FACS buffer (2% FCS [HyClone], in PBS [Gibco]). Analyses were performed using FlowJo and data statistically tested using GraphPad Prism™ software, with specific statistical tests described in respective figure legends.

Flow cytometry was performed on single cell suspensions using these mouse antibodies; PerCp-CD4, Alexa-488-CD4, Alexa-647-FoxP3, PE-IL17A, FITC-CD25, PE-PD1 (Biolegend), PE-FoxP3, PC-IL17, FITC-CD25, APC-CD11b, FITC-PDL2, PE-PDL1 (eBiosciences). Flow cytometry was run on an Accuri benchtop C6 cytometer and analysed using FlowJo™ software. For all analyses unless otherwise stated, samples were gated on lymphocyte populations based on size, as assessed by SSC and FSC, followed by gating for CD4 positivity. Intracellular staining was performed by fixation and permeabilization using eBioscience Fix-Perm Intracellular staining buffers (Cat. Num 8222-49 and 8333-56). Staining was performed for 1 hour at room temperature followed by washing in FACS buffer (2% FCS (Hyclone), in PBS (Gibco)) Analyses were performed using FlowJo™ and data statistically tested using GraphPad Prism™ software, with tests described in respective figure legends.