Mouse anti hnrnp a1 clone 4b10

Animals sacrificed for immunofluorescent analysis were perfuse fixed with 4% paraformaldehyde. Following a 48-h post-fixation period, tissues were put through a sucrose gradient and embedded in OCT. Sections were cut at 10 μm and stored at −80 °C until further use. For staining, sections were thawed at room temperature followed by PBS washing. Sections were blocked using 100% SeaBlock Blocking Solution (ThermoFisher) for 1 h at room temperature before being placed into primary antibody and incubated overnight at 4 °C. Secondary antibodies were incubated with sections for 1 h at room temperature the following day and then mounted using ProLong Gold. The following antibodies were used: mouse anti-hnRNP A1 (clone 4B10; Millipore) and donkey anti-mouse Alexa Fluor 488 (Jackson Immunoresearch).

The following was carried out with RNase-free reagents, under RNase-free conditions, on ice unless otherwise noted, and is based upon the eCLIP RNAseq method and buffers described by van Nostrand et al.^{49 (link)}. In brief, spinal cord fragments were pelleted and re-suspended in iCLIP lysis buffer^{49 (link)} containing RNase and protease inhibitors, then homogenized using a Tissue Tearor homogenizer and incubated on ice for 30 min to complete lysis. After pelleting tissue debris, supernatant was collected and assessed for protein concentration, and 2 mg protein per sample was prepared for immunoprecipitation with 50 µL Pierce magnetic protein A/G beads (Fisher Scientific Canada PI88803) and 2 µg mouse anti-hnRNP A1 (clone 4B10, Millipore Canada). 2 mg pooled naïve and pooled EAE samples were also prepared for immunoprecipitation with 2 µg of a control mouse IgG2b antibody (clone GC198, Millipore Canada). Samples were treated with Turbo DNAse (Ambion; Fisher Scientific Canada) and RNase I (Fisher Scientific Canada) at a concentration of 1:250 (Supplementary Fig. 10) for 5 min at 37 °C, then incubated with beads and antibody overnight with rotation at 4 °C.