Anti rabbit and anti mouse igg peroxidase conjugated secondary antibodies

The following primary antibodies were used for immunofluorescence: anti-rabbit S100B (1 : 7500, Novus Biological), anti-mouse S100B (1 : 1000, Sigma Aldrich), anti-mouse RAGE (1 : 200, Millipore), anti-rabbit GFAP (1 : 1000, Dako), anti-mouse GFAP (1 : 1000, Novus Biologicals), anti-mouse NeuN (1 : 500, Millipore), anti-rabbit Iba1 (1 : 200, Wako), anti-mouse CNPase (1 : 500, Novus Biologicals), and anti-rabbit ChAT (1 : 200, Millipore). Secondary fluorescent antibodies were Cy3 Donkey anti-rabbit (1 : 200), Alexa-Fluor 488 Donkey anti-rabbit (1 : 200), Cy3 Donkey anti-mouse (1 : 200), and Alexa Fluor 488 Donkey anti-mouse (1 : 200) from Jackson ImmunoResearch Laboratories. To-Pro-3 (1 : 10,000, Thermo Fisher Scientific) was used to stain nuclei. The primary antibodies used for Western blotting were anti-S100B (1 : 1000, Novus Biologicals), anti-rabbit RAGE (1 : 1000, Thermo Scientific), anti-mouse RAGE (1 : 1000, Millipore), anti-GAPDH (1 : 10,000, Millipore), anti-SOD1 (1 : 1000, Enzo Life Sciences), and anti-GFAP (1 : 5000, Novus biologicals). Anti-rabbit and anti-mouse IgG peroxidase-conjugated secondary antibodies (1 : 2500) were from Bio-Rad Laboratories.

The following primary antibodies were used for immunofluorescence (IF) or western blot (WB): anti-rabbit S100A4 (1:500-IF, 1:1000-WB, Millipore, Burlington, MA, USA), anti-mouse glial fibrillary acidic protein (GFAP) (1:1000-IF, Novus Biologicals, Centennial, CO, USA), anti-rat CD68 (1:500-IF, Abd Serotec, Kidlington, UK), anti-rat CD11b (1:500-IF, Abd Serotec), anti-mouse paxillin (1:500-IF, 1:1000-WB, BD-Biosciences, San Jose, CA, USA), anti-mouse gp91^phox (1:1000-WB, BD-Biosciences), anti-rabbit mTOR and phospho-mTOR (1:1000-WB, Cell Signaling, Danvers, MA, USA), anti-rabbit NF-κB and phospho-NF-κB (1:1000-WB, Cell Signaling), anti-GAPDH (1:5000-WB, Millipore). Secondary fluorescent antibodies for IF were: Cy3 Donkey anti-rabbit (1:200), Alexa-Fluor 488 Donkey anti-rabbit (1:200), Cy3 Donkey anti-mouse (1:200), and Cy5 Donkey anti-rat (1:200) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Phalloidin (1:200, Sigma Aldrich, Milan, Italy) was used to stain cells’ actin filaments. DAPI (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) was used to stain nuclei. Anti-rabbit and anti-mouse IgG peroxidase-conjugated secondary antibodies (1:2500) were from Bio-Rad Laboratories (Hercules, CA, USA).

Spinal cords of at least 5 animals per genotype were dissected and lysed in RIPA buffer (50 mM Tris HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1% Triton X-100, 0.25% Na-Deoxycholate, 0.1% SDS, protease inhibitor cocktail). After 2 × 10″ sonication cycles, samples were incubated on ice and then centrifuged at 18000 × g for 20′ at 4 °C. Supernatants were then quantified with Bradford protein assay (Bio-rad) and resuspended in Laemmli Buffer before SDS-PAGE (Sigma-Aldricht). Antibodies used: rabbit anti-FUS (Bethyl), mouse anti-GFAP (Cell Signaling), mouse anti-Iba1 (Wako), rabbit anti-Gemin2 (Proteintech), mouse anti-Sm B/B’ (Thermo Scientific), mouse anti-SMN (BD), mouse anti-β-actin (Sigma).Anti‐rabbit and anti‐mouse IgG peroxidase‐conjugated secondary antibodies were from Bio Rad.