Anti β tubulin 3

Primary cortical neurons were isolated from the cerebral cortex of E16 embryonic C57BL/6 mouse pups according to previously described methods (Kim and Magrane, 2011 (link)). The cortices were dissected out. After enzyme dissection, the extracted cortical neurons were counted and plated on sterilized cover glass in six-well plates in serum-free neurobasal medium supplemented with appropriate growth supplements (B27 and N2, Life Technologies, Carlsbad, CA, United States) to remove astrocytes, oligodendrocytes and microglia. Primary cortical neurons were cultured with different concentrations of myelin debris for 48 h. Cells were fixed and stained with anti-β-tubulin III (1/400, Abcam). Stained images were visualized using a fluorescence microscope (Carl Zeiss Axio Observer Z1, Oberkochen, Germany), and axon length was measured using ImageJ software (National Institutes of Health, Bethesda, MD, United States) in a blinded manner.

The HSCs were grown on different conduits for 7 days. Then, the conduits with cells were rinsed using PBS and subsequently fixed by placing them in 4% paraformaldehyde (Sigma-Aldrich) for 20 min at normal room temperature. After this, the cells were permeabilized using 0.1% PBS-T, immune-stained with anti-β-tubulin III (Abcam), and then with anti-mouse conjugated tetramethylrhodamine (TRITC, Invitrogen). The phalloidin affixed with Alexa Fluor 488 (1:300 dilution in PBS, Invitrogen) was used to stain the intracellular F-actin filaments. The immunofluorescence staining results of the HSCs on the conduits were observed under a Leica TCS SP8 X white light laser confocal microscope (Leica Microsystems GmbH, Wetzlar, Hessen, Germany).

Spinal cord and bladder tissues were embedded in optimum cutting temperature compound. Sections were prepared at 5 μm thicknesses and stained with HE for spinal cord tissues and HE and Mason’s trichrome for bladder tissues. Anti-β-tubulin III (Abcam, Cambridge, UK) and anti-GFAP (Abcam) antibodies were used for the immunofluorescence staining of the repaired spinal cords. The positive staining areas of β-tubulin III and GFAP were analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). Three slides of each animal were counted in each group. Immunofluorescence staining with antibodies against p63, krt5, krt20, collagen I, α-smooth muscle actin (α-SMA, Abcam), and Upk (Santa Cruz Biotechnology, Dallas, TX, USA) was performed to examine remodeling of the urothelium, smooth muscle cells, and collagen in the bladder wall.

For BMDMs and cortical neuron coculture, we used a Transwell system in which cortical neurons were seeded in the lower chamber and BMDMs precultured with myelin debris were seeded in the upper chamber. The myelin debris-treated BMDMs in the upper chamber were divided into two groups, namely, with or without BMSC-Exos administration. After 48 h of coculture, the cortical neuron cells in the lower chamber were fixed and stained with anti-β-tubulin III (1:400, Abcam) for axon visualization. The axon length was measured using ImageJ software (National Institutes of Health, United States).

After culturing the cells as described above, were fixed in 4% PFA in PBS for 15 min and permeabilized in CH₃OH for 7 minutes at −20° C. Cells were incubated in 4% BSA (Sigma Aldrich, USA) for 30 minutes then with the subsequent primary overnight at 4° C: rabbit polyclonal anti-β-Tubulin III (1:1000 Abcam, Cambridge, UK), rabbit polyclonal anti-TH (1:200, Novus Biologicals, Centennial, USA), mouse monoclonal anti-GAP43 (1:200, Abcam, Cambridge, UK). After several washings, coverslips were incubated with secondary antibodies, goat anti-mouse or anti-goat IgG Alexafluor 488 or 633 or 546 (1:2000 Life Technologies, California, USA), for 1h at RT. After different washes, Vectashield mounting with DAPI (Vector Laboratories Burlingame, USA) were used. All the samples were observed using confocal laser microscope (Leica, Wetzlar, Germany).

Immunocytochemistry was performed using an immunostaining protocol as described previously [17 , 21 ]. The primary antibodies were anti-β-tubulin III and anti-GFAP (1:200; Abcam, UK) incubated with the cells overnight at 4 °C. Hoechst 33342 (10 μM) was used to stain nuclei. Images were acquired as Z-stacks (typically of ~10-μm focal depth) using a Zeiss 710 VIS CLMS confocal microscope. For the quantification of immunofluorescence, Z-stack images were analysed with freely available ImageJ software (NIH, Bethesda, USA). For this, images were processed in 2D mode to separate the GFAP fluorescent signal (green channel) and β-tubulin III immunofluorescence (red channel), the total area of each signal was quantified using an ImageJ plugin for particles/area analysis and normalized to the cell density (nuclei staining). Six neuronal cultures were examined per group.

Induced osteogenic cells were fixed by 4 % paraformaldehyde (Sigma-Aldrich) and stained by anti-osteocalcin antibody (1:200 dilution; Santa Cruz); induced neurogenic cells were stained with anti-GFAP antibody (1:300 dilution; CST), anti-Nestin antibody (1:300 dilution; CST), anti-MAP2 antibody (1:200 dilution; Abcam), and anti-β-Tubulin III (1:200 dilution; Abcam); and induced pancreatic islet cells were stained with anti-Pdx1 antibody (1:400 dilution; CST), anti-glucagon antibody (1:400 dilution; CST), and anti-insulin antibody (1:400 dilution, 3014; CST) respectively. Total RNA was extracted using TRIZol® Reagent (Life Technologies) followed by cDNA synthesis using M-MuLV Reverse Transcriptase and Oligo (dT)23VN (NEB). qPCR was performed with iTaq™ Universal SYBR® Green Supermix (Bio Rad). Western blotting was performed for osteocalcin, microtubule-associated protein 2 (MAP2), β-Tubulin III, ADFP, and Plascidin L. All antibodies were validated for antigen specificity.