Rabbit anti βiii tubulin

miR-2110 mimic, siRNAs and negative control oligos were purchased from Dharmacon. Rabbit anti-βIII-tubulin, anti-GAP43, anti-NSE, anti-TSKU, anti-calnexin, and HRP-conjugated anti-rabbit IgG antibodies were obtained from Thermo Fisher Scientific. Anti-cleaved PARP was purchased from Cell Signaling Technology. BE(2)-C, SKNDZ, CHLA-90, and SKNFI cells were from the American Type Culture Collection (ATCC). Kelly cells were obtained from the cell line repository at the Greehey Children’s Cancer Research Institute at the University of Texas Health San Antonio. Cells were grown in DMEM/F12 (Corning Cellgro) supplemented with 10% Equafetal bovine serum (Atlas Biologicals).

For tissue immunofluorescence, mice were transcardially perfused with ice-cold PBS, and lumbar spinal cords and L3-L4-L5 DRGs were harvested and placed into 4% paraformaldehyde (PFA) (Sigma-Aldrich). Transverse sections of the spinal cord (20 μm) and DRG (10 μm) were taken using a cryostat (Bright Instruments). Sections were postfixed for 10 minutes, permeabilized with PBS/0.1% Triton X-100, and then incubated with rat anti–mouse F4/80 (1:200; Abcam, ab6640), followed by anti-rabbit–Alexa Fluor 546 secondary antibody (1:1000; Invitrogen, A11081), Ki67 (1:250; Abcam, ab16667), CCL2 (1:500; Invitrogen, MAS-17040), and IB4 (1:500; Invitrogen, I32450). For macrophage and DRG culture immunofluorescence, cells were plated on Labtec chamber slides (Thermo Fisher Scientific), fixed with 4% PFA, and then permeabilized with PBS/0.1% Triton X-100 for 10 minutes. The following antibodies were used: rabbit anti–mouse p-SMAD2/3 (1:1000; Cell Signaling Technology, 8828), rat anti–mouse F4/80 (1:200; Abcam, ab6640), rabbit anti–mouse TGF-βR2 (1:100; R&D Systems, FAB532P), rabbit anti–β-III tubulin (1:1000; Thermo Fisher Scientific, MA1-118), followed by fluorophore-coupled secondary antibodies (1:1000, Alexa Fluor 568, 488, 647; Invitrogen). The immunoreactivity was captured using a Zeiss LSM710 confocal microscope and images were acquired using the LSM software (Zeiss).