Alexa 488 conjugated goat anti rabbit antibody

The lumbosacral enlargement was collected in mice of CON group and SR group, fixed in 4% paraformaldehyde, and dehydrated in 30% sucrose. The third to fifth lumbar vertebrae (L3–L5) were transversely cut into 25-μm-thick sections and mounted on glass slides. Sections were blocked and then incubated with primary antibodies against Iba-1 (1:700, Wako, Tokyo, Japan) and GFAP (1:500, Abcam, Cambridge, UK) or CD31 (1:500, Abcam, Cambridge, UK) overnight at 4°C. After washing with PBS, the sections were incubated with Alexa 488-conjugated goat anti-rabbit antibody (1:1000, Thermo Fisher Scientific, MA, USA) and Alexa 594-conjugated goat anti-mouse antibody (1:1000, Thermo Fisher Scientific, MA, USA) in the dark. Three spinal cord sections per animal were used for statistical analysis. The fluorescence intensities of Iba1, GFAP, and CD31 were measured using ImageJ software (NIH, Bethesda, MD, USA).

Antibodies used for immunoblotting were anti-SRSF1 (1:1000, Invitrogen, 32–4500), anti-Rbfox1 (1:100, N-14, Santa Cruz Biotechnology, sc-135476), anti-Rbfox2 (1:2000, Bethyl Laboratories, A300-864A), anti-hnRNP F/H (1:500, Santa Cruz Biotechnology, sc-32310), anti-U1-70K (1:1000, Synaptic Systems, 203011), anti-U1A (1:1000, Thermo Fisher Scientific, PA5-27474), anti-U1C (1:1000, Sigma-Aldrich, SAB4200188), anti-GAPDH (1:2500, Sigma-Aldrich, G9545), anti-6xHis-tag (1:2500, Medical & Biological Laboratories, D291-3), anti-Flag M2 (1:1000, Sigma-Aldrich, F3165), anti-U2AF65 (1:400, MC3, Santa Cruz Biotechnology, sc-53942), anti-GFPT1 (1:500, Abcam, ab125069), anti-RL2 (1:800, Santa Cruz Biotechnology, sc-59624), anti-O-GlcNAc (CTD110.6) (1:2000, Cell Signaling Technology, 9875), anti-β-actin (1:1000, Santa Cruz Biotechnology, sc-47778), and anti-synaptophysin antibodies (1:50, Innovative Research, 18–0130). The secondary antibodies were anti-mouse IgG (1:2000, Cell Signaling Technology, 7076) conjugated to horseradish peroxidase, goat anti-rabbit IgG (1:2000, Cell Signaling Technology, 7074) conjugated to horseradish peroxidase, and Alexa 488-conjugated goat anti-rabbit antibody (1:1000, Thermo Fisher Scientific, A-11034). AChRs were visualized by Alexa 594-conjugated ɑ-bungarotoxin (1:1000, Invitrogen, B-13423).

After general anesthesia was administered, mice were intracardially perfused with normal saline followed by 4% paraformaldehyde. The lumbar enlargements were removed, post-fixed with 4% paraformaldehyde and then dehydrated in 30% sucrose. Samples were cut into 20 µm sections using a freezing microtome. After washes with phosphate buffer saline (PBS), the sections were blocked with 10% goat serum containing 0.3% Triton X-100 and then incubated with the following primary antibodies in 10% goat serum overnight at 4℃: Iba1 (rabbit, 1:500, Wako, Japan), GFAP (mouse, 1:500, CST, USA), CB2R (rabbit, 1:100, Abcam, USA) and OX42 (mouse, 1:500, Abcam, USA). After washes with PBS, sections were incubated with secondary antibodies in 10% goat serum, including an Alexa 488-conjugated goat anti-rabbit antibody (1:3000, Thermo Fisher, USA) and Alexa 594-conjugated goat anti-mouse antibody (1:3000, Thermo Fisher, USA). Then, sections were washed with PBS, transferred to glass slides, air-dried and stained with 4’,6-Diamidino-2-Phenylindole (DAPI) (Abcam, USA). Images were captured using a laser-scanning confocal microscope (Olympus, Japan). Three sections from three mice from each group were analyzed.

RVECs seeded in 24-well plates were pretreated with/without HCQ for 10 h and incubated with TNF-α for another 24 h at 80%–90% confluency. After stimulation, cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, permeabilized with 0.3% Triton X-100 for 20 min, and blocked with 3% BSA for 2 h at room temperature. Subsequently, they were stained overnight with rabbit anti-phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, Danvers, Massachusetts, USA) at 4°C, washed with PBS, incubated with Alexa 488-conjugated goat anti-rabbit antibody (Invitrogen, California, USA) for 2 h, and then with DAPI (Abcam, Cambridge, UK) for 10 min. Cells were imaged using an inverted fluorescent microscope (Nikon).

The frozen brain was sectioned (20 μm thickness). Sections were fixed with 4% paraformaldehyde and stained with a rat anti-mouse CD68 antibody (Cat^# ab955, Abcam, Waltham, MA, United States) or rabbit anti-mouse iNOS (Cat^# ab15323, Abcam) (all 1:100) followed by incubation with Alexa 488-conjugated goat anti-rat antibody (Cat^# A-11001) or Alexa 488-conjugated goat anti-rabbit antibody (Cat^# A11008) (All from Invitrogen, Waltham, MA, United States). Sections were counterstained with DAPI and mounted for imaging acquisition.

For intracellular staining, mouse BM was first stained with HSC surface markers, as described above. Peripheral blood from NSGS mice was lysed with ammonium chloride potassium (ACK) buffer, and remaining MNC cells were washed and fixed with 4% paraformaldehyde in the dark for 15 minutes and permeabilized with 0.5% Saponin permeabilization solution (BD Bioscience) for 1 hour at room temperature. After washing, cells were labeled with rabbit anti-5hmC antibody (active motif, 1/200, 30 mins at 4 °C) or isotype control and then incubated in Alexa 488-conjugated goat anti-rabbit antibody (Invitrogen, 1/1000, 30 mins at 4 °C), washed and analyzed by flow cytometry.