Dmx 1200f

Embryoid bodies-hGMSCs were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 1 h at room temperature (RT). After fixation, the samples were post-fixed with 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Epon. Semithin sections were stained with toluidine blue and used for light microscopy analysis. The sections were observed with a Zeiss Axiophot apparatus, and images were captured using a Nikon digital camera Digital Sight (Diomede et al., 2016b (link)). Thin sections were stained with uranyl acetate and lead citrate; some sections were also stained with tannic acid, and finally observed with a Zeiss EM 109 transmission electron microscope. Images were captured using a Nikon digital camera Dmx 1200F and ACT-1 software.

Cells were fixed with 4% paraformaldehyde in PBS, pH 7.4 at the end of proliferation and differentiation stages. Immunocytochemistry assays were performed following standard protocols [57 (link)]. Briefly, cells were permeabilized and blocked for 1 hour with 0.3% Triton X-100 and 10% normal goat serum (NGS) in PBS. Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore). Appropriate Alexa-Fluor secondary antibodies were used diluted in PBS (1:1000; Molecular Probes) containing 10% NGS. Nuclei were stained with Hoechst 33258 (1 ng/mL; Sigma). Immunostainings were analyzed with an epifluorescence microscope (Nikon, Eclipse TE2000-U) and photographed with a Nikon digital camera (DMX1200 F). Negative controls were performed in the absence of primary antibodies and showed no unspecific staining.

iPSC colonies were cultured in 24-well plates with glass coverslips pre-coated with 0.5% gelatin. Subsequently, the culture medium was removed, cells were washed with PBS three times, and fixed with 4% paraformaldehyde for 10 min. Cells were permeabilized and blocked for 1 h in PBS with 0.3% Triton X-100 and 10% normal goat serum. Then, incubation with primary antibody diluted in blocking solution (PBS with 10% of normal goat serum) was done overnight at 4 °C. After washing, cells were incubated with appropriate secondary antibodies for 1 h at room temperature, counterstained with DAPI, and mounted with Aqua-Poly/Mount (Polysciences, Warrington, PA, USA). The following antibodies were used at the indicated dilutions: mouse anti-OCT4 (BD Biosciences 611202, 1:250), rabbit anti-SOX2 (Sigma-Aldrich AB5603, 1:500), and rabbit anti-NANOG (Prepotech 500-P236, 1:1000). As secondary antibodies, goat anti-mouse conjugated with Alexa Flour 568 (Thermo Fisher Scientific) and goat anti-rabbit conjugated with Alexa Flour 647 (Thermo Fisher Scientific) were used according to the supplier’s instructions at a dilution of 1:500. Immunostainings were analyzed with an epifluorescence microscope (Nikon, Eclipse TE2000-U) and photographed with a Nikon digital camera (DMX1200 F).

6647 and LAP-35 untreated or treated cells were fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4, post-fixed with 1% OsO4, dehydrated to absolute ethanol and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate before observation with a Zeiss EM109 electron microscope. Images were captured using a Nikon digital camera Dmx1200F and ACT-1 software.