M1 embedding matrix

Eight weeks after transplantation, animals were deeply anesthetized with 5% inhaled isoflurane and transcardially perfused with ice cold 0.1M PBS followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). A 2cm section of cervical cord centered on the lesion was harvested and post-fixed in 4% PFA overnight at 4°C followed by 48hrs of cryoprotection in 30% sucrose in PBS at 4°C. The sample was then snap frozen in M1 Embedding Matrix (Thermo Fisher Scientific, Waltham, MA) on dry ice. Serial 35μm thick cryosections were then arranged on Superfrost glass slides (Thermo Fisher Scientific) as either six cross-sectional slices or two longitudinal slices.

On the 13th day of culture when MSCs reached 80% confluency, CPN was added to MesenCult media in 1 : 4 ratio (25 μg/mL). After MSCs were incubated with CPN for 24 h, MSCs were centrifuged for 5 min at 1500 rpm in round bottom tubes to form cell spheres. The cell spheres were induced via specific chondrogenesis induction medium composed of 10 ng/mL TGFβ (transforming growth factor beta) 1, 100 nM dexamethasone, 50 μg/mL ascorbic acid, 1 mM sodium pyruvate, 6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 μg/mL selenous acid (ITS), and 1.25 mg/mL bovine serum albumin. Cell spheres were cultured for 28 days in a 5% CO₂ incubator at 37°C. The MSCs were not further treated with CPN during differentiation. To assess the effect of CPN on chondrogenic differentiation, the cell nodules were fixed with M1 Embedding Matrix (Thermo) and Alcian Blue (Sigma) was used to detect the presence of cartilage condensations [23 (link)]. The cells were then examined under bright-field microscopy. For CPN detection, the same sections were examined under fluorescence microscopy with FITC filter. For the evaluation, we used an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm. For counter staining to visualize nuclei, the specimens were mounted using UltraCruz (Santa Cruz) mounting medium with DAPI.

At the survival endpoint of 7 days after SCI contusion or immediately for naïve, the animals were perfused. The rats were euthanized by carbon dioxide inhalation and then subjected to transcardial perfusion. After perfusion with 500 mL cold (4 °C) physiological saline, animals were perfused with 400 mL cold phosphate-buffered 4% paraformaldehyde (0.1M, pH 7.4). The CNS tissue was extracted and underwent overnight post-fixation in 4% paraformaldehyde, followed by cryoprotection in sucrose according to the previously described procedures [38 (link),40 (link)]. A 2 cm segment of the thoracic T7-9 spinal cord, encompassing the injury epicenter, was embedded in M-1 embedding matrix (ThermoScientific, Kalamazoo, MI, USA) for cryosectioning into 20 series at a thickness of 20 μm (coronal) using a Leica CM3050S Cryostat (Leica Microsystems Inc., Buffalo Grove, IL, USA). The tissue sections were stored at −20 °C until further processing.

Human brain samples and mouse brains were washed in PBS and cryo-preserved in 30% sucrose in PBS for 36 h approximately. Both mouse and human samples were embedded in M-1 embedding matrix (Thermo scientific, USA) or Optimal Cutting Temperature (OCT) compound (Tissue-Tek), and cut into 50 μm to 100 μm -thick slices on a sliding freezing microtome (human) or cryostat (mouse) and kept at −20 °C in a cryoprotectant solution containing ethylene glycol (30%), and glycerol (30%) in 0.05 M phosphate buffer (PB, pH 7.4) until processed for immunofluorescence.