Ab18207

Mouse mesenchymal stem cells (MSC) were cultured as previously described [20 (link),21 (link),22 (link)] in a culture medium containing low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine and 1% non-essential amino acids. Primary mouse neural stem cells (NSCs) were seeded onto Geltrex coated 96-well plates and differentiated as previously described [23 (link)]. Cell phenotype was confirmed by immunohistochemistry as previously described [20 (link),23 (link)] using primary antibodies for mouse βIII-Tubulin (1:1000 dilution, Ab18207, Abcam, Cambridge, UK) and mouse GFAP (1:250 dilution, MA515086) and secondary antibodies Alexa Fluor 488 anti-rabbit (1:500 dilution, A11008) and Alexa Fluor 594 (1:500 dilution, A11001), using a Zeiss Elyra PS.1 microscope (ZEISS, Cambridge, UK) equipped with C-Apochromat 63×/1.2 W Korr M27 objective for confocal imaging followed by ImageJ thresholding.

For fluorescent microscopy, the cultured cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilised with 0.1% Triton X-100 for 10 min. After blocking for 30 min with 5% bovine serum albumin, the washed cells were incubated for 1 h at room temperature or overnight at 4°C with a primary antibody against the POU domain, class 5, transcription factor 1 (OCT4) (1:200, ab19857, Abcam), Transcription factor SOX-2 (SOX2) (1:200, ab92494, Abcam), Homeobox protein NANOG (NANOG) (1:100, ab109250, Abcam), Tra-1-60 (1:150, MAB4360, Merck), Tubulin β-3 (TUBB3) (1:500, ab18207, Abcam), smooth muscle alpha actin (SMA) (1:400, A2547, Merck) or α-fetoprotein (AFP) (1:200, ab3980, Abcam). Subsequently, the cells were incubated with fluorescence-labelled secondary Alexa Fluor 594 or 488 (1:1000, A11001, A11008 and A11012, Invitrogen) at room temperature for 1 h, protected from light. The cells were further incubated with 1 µM DAPI for nuclear staining. Between incubations, samples were washed with PBS containing 0.1% Triton X-100. Alkaline Phosphatase Live Stain 500X from Thermo Fisher Scientific was used for determining the enzyme activity following the manufacturer's instructions. Pictures were acquired using a Floyd Cell imaging station (Life Technologies).

35 μm sagittal tongue sections from the tongue midline were washed for 5 min at RT in TBS (42 mM Tris HCl, 8 mM Tris Base, 154 mM NaCl; pH 7.4). Sections were blocked in 20% normal donkey serum (NDS) in TBS-T for 1 hr at RT and incubated overnight at 4 °C in primary antibody diluted in TBS-T +2% NDS/NGS. Primary antibodies against CD31 (Millipore MAB13982; 1:200), CD68 (Biolegend 137002; 1:100), GFP (AbCam; ab13970; 1:200), LYVE1 (ReliaTech 103-PA50AG; 1:500), Podoplanin (Biolegend 156202; 1:50), TUJ1 (AbCam ab18207; 1:200) were used. Sections were then washed 3 × 30 min in TBS-T and incubated overnight at 4 °C with appropriate secondary antibodies raised in goat and conjugated to Alexa Fluor dyes (Invitrogen) were diluted 1:500 or 1:1,000 in TBS-T +2% NDS/NGS. Sections were washed 3 × 30 min in TBS-T and nuclei were stained with 0.5 mg/ml DAPI in TBS. After washing, sections were mounted with FluorSave reagent (Calbiochem).

RGCs were harvested and seeded in a 24-well culture plate (3 × 10⁴ cells/well). After incubation for 24 h, 48 h, and 72 h, cells were fixed with 4% paraformaldehyde at RT for 30 min according to previously described methods [15 (link)–17 (link)]. After washing with Hank's Balanced Salt Solution (HBSS basic 1x, Gibco Company, USA), cells were permeabilized in 0.3% Triton (Sigma-Aldrich, Germany) in HBSS for 15 min at RT. The cells were then blocked with a blocking buffer containing 1% bovine serum albumin (BSA) and 5% goat serum in HBSS for 60 minutes after washing with HBSS. After incubation with the primary antibodies, rabbit anti-beta III tubulin (1 : 1000,Cat#ab18207,Abcam,Cambridge, MA), which was specific for neurofilament heavy chains [18 (link)], and mouse anti-Brn3a (1 : 25, Cat#sc-8429, Santa, USA), which was specific expression in RGCs nucleus [17 (link), 19 (link)] for overnight at 4°C, the cells were washed three times and incubated with secondary antibodies (goat anti-mouse and goat anti-rabbit) (1 : 500, Cat #ab150077, Cat #ab150116, Abcam, Cambridge, MA) at RT for 2 hours with light-shielded and counterstained with DAPI. Five fields were randomly photographed under a 200-fold inverted microscope (Carl Zeiss, Jena, Germany). The number of cells which had neurite lengths equal to or greater than three times the cell body diameter was calculated by ImageJ.

Briefly, hiNSC were grown in coated 96-well culture plates, fixed in 4% paraformaldehyde, washed 3 times in 1X phosphate-buffered saline (PBS), blocked in PBS with 10% goat serum and 0.1% triton X-100 for 1 h at room temperature (RT), incubated in primary antibody diluted in blocking buffer overnight at 4 °C, the following day rinsed in PBS 4 times and incubated in the corresponding fluorescently conjugated secondary antibody in blocking buffer for 1 h at RT protected from light, and finally we counterstained the nuclei with DAPI or Hoechst for 5 min at RT. Primary antibodies used were rabbit anti-βIII-tubulin (TUJ1) 1:500 (abcam-ab18207), rabbit anti-choline acetyl transferase (ChAT) 1:500 (Thermo Fisher-50-173-3063), rabbit anti-serotonin reuptake transporter (SERT) 1:500 (abcam-ab272912), and rabbit anti-glutamate decarboxylase 67 (GAD67) 1:500 (Thermo Fisher-PA5-21397). The secondary antibody used was goat anti-rabbit Alexa 488 conjugated 1:1000 (Thermo Fisher-A-11070). Imaging was done using an EVOS M7000 System (Thermo Fisher).