Ab52642

The cells were detached with Versene solution (PanEco, Moscow, Russia) and pelleted by centrifugation at 1800 rpm for 5 min, then washed with HBSS (PanEco, Moscow, Russia), fixed in 4% paraformaldehyde for 10 min, washed with PBS, permeabilized with 70% methanol on ice for 10 min, washed twice with PBS and collected by centrifugation at 1800 pm for 5 min. Fixed cells were incubated with primary antibodies to PAX6 (ab5790, Abcam, Cambridge, UK), glial marker S100b (ab52642, Abcam, Cambridge, UK) or neuronal marker βIII tubulin (ab182070, Abcam, Cambridge, UK) at +4 °C for 12 h; washed with PBS; collected by centrifugation at 1800 rpm for 5 min; and incubated with secondary antibodies (Alexa Fluor 488 conjugated, Invitrogen, Carlsbad, CA, USA) for 60 min in the dark. Fixed cells exposed to secondary antibodies were only used as a negative control for the flow cytometry-based quantification. Stained cells were analyzed on a CyFlow ML flow cytometer using the FloMax software (Partec, Goerlitz, Germany). For evaluation of the number of immunopositive cells, the experiment was repeated at least 3 times.

Cultured astrocytes were lysed in RIPA buffer with 13 complete protease inhibitors (Roche). Protein levels were assessed with a Bradford assay with BSA as the standard. Approximately 10 µg of denatured proteins was separated by 8% SDS-polyacrylamide gel electrophoresis and blotted onto 0.22 µm PVDF membranes (Roche). Nonspecific binding was blocked with TBST (TBS-0.1% Tween-20) with 3% (w/v) nonfat milk for 2 h at room temperature. Membranes were incubated overnight at 4℃ in TBST with 5% milk and the following primary antibodies: rabbit anti-GFAP (1:1000, 20,044,021, Dako), rabbit anti-MAP2 (1:500, 17,490–1-AP, Proteintech), rabbit anti-S100 beta (1:1000, ab52642, Abcam), and mouse anti-β-actin (1:5000, 60,008–1-IG, Proteintech). Membranes were then incubated at room temperature for 2 h in TBST with 5% milk and secondary antibodies (1:5000, Invitrogen). Protein bands were detected by chemiluminescence (Tanon, Shanghai, China) and quantified by densitometry with ImageJ (ImageJ 7.0 software). Protein levels were normalized to the level of β-actin as a control.

Masseter muscles were dissected and stored in 30% sucrose/PBS ON at 4°C. Following, the tissue was embedded in Tissue-Tek O.C.T. (optimal cutting temperature) compound and frozen by nitrogen-cooled isopentane. Embedded sections were cut into 10 μm thick sections with a cryotome. Prior immunohistochemistry, the sections were post-fixed in 4% PFA for 10 min. For primary antibodies, anti-βIII-tubulin (mouse, 1:2000, Covance, MMS-435P), anti-S100β (rabbit, 1:1000, Abcam, ab52642), Vimentin (rabbit, 1:130, Abcam, ab 7783) and CD45 (rat, 1:1000, BD Pharmingen, 550539) were used. For detection of the primary antibodies, Alexa Fluor 488- or 546 conjugated secondary antibodies were used.

Adipose-derived stem cells were seeded on coverslips covered with poly-L-lysine (No. P8920; Sigma) in 6-well plates and the immunofluorescence method was applied to show SC associated biomarkers.
After incubation for 24 h, the medium in the wells was removed. Coverslips with attached cells were then washed with 1 × PBS. The cells were then fixed with 4% paraformaldehyde for 10 min. After washing three times with PBS for 5 min each time, ADSCs were blocked with 10% goat serum (SL038; Solarbio) in PBS for 1 h. Primary antibody anti-S100β (1:200, ab52642, Abcam, United States); It was dropped onto coverslips and incubated at room temperature overnight. Next, secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594; 1:200; ab150084; Abcam), was dropped onto coverslips. After incubation with secondary antibodies in the dark for 1.5 h, the coverslips were washed and incubated with DAPI (No. H-1200; Vector Laboratories Inc., Burlingame, CA, United States). For the final step, the coverslips were inverted onto the slides and analyzed under a fluorescent microscope. The number of surviving cells, and the rates of S100β - positive cells in the 5 groups of ADSCs were calculated according to 5 randomly selected fields in each group at 200 × magnification using IPP 6.0.

Mice were anesthetized and then perfused with PBS and 4% paraformaldehyde (PFA). Brain was dissected and fixed in 4% PFA at 4°C overnight. Then the brain was dehydrated with 10%, 20% and 30% sucrose in order. The dehydrated brain was mounted with O.C.T. compound and sectioned at 30 mm. After washed by PBS (three times, 15 min per time), the collected brain slices were permeabilized with 0.5% Triton‐X100 for 30 min at room temperature and the blocked with blocking buffer for 1 h. Next, the diluted primary antibodies (S100b, 1:200, ab52642, Abcam, UK; Wnt9a, 1:1000, ab125957, Abcam, UK; Wnt10a, 1:1000, ab106522, UK) were applied on brain slices at 4°C overnight. After incubation, brain slices were washed by PBS (four times, 10 min per time), and incubated with the secondary antibodies (Donkey anti rabbit 488, 1:500, 711‐545‐152, Jackson ImmunoResearch Laboratories Inc., US or Donkey anti rabbit Alexa Fluor™ 488, 1:500, A‐21206, ThermoFisher Scientific) for 2 h at room temperature, and then with DAPI for 5 min at room temperature. Brain slices were washed by PBS (four times, 10 min per time) and mounted with AQUA‐MOUNT (Thermo Scientific, US). Brain slices were scanned by VS200 (Olympus, Japan) and fluorescence imaging microscope (Eclipse Ni, Nikon Inc, Japan) and confocal microscope (LSM 880, ZEISS, Germany).