Mouse anti ssea 4

For immuno-staining, cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. Non-specific binding was blocked with 5% goat serum in PBS. The following antibodies were applied on each well independently: mouse monoclonal primary antibody against human microtubule associated protein-2 (MAP-2) or against human βIII-tubulin, rabbit anti-human tyrosine hydroxylase (a gift from Dr. Yves Sauve, University of Alberta), rabbit anti-glial fibrillary acidic protein (GFAP, 1:500), rabbit monoclonal against S100B (1:100; abcam), mouse anti-SSEA-4 (10 μg/ml; Millipore), mouse anti-human CD31 (1 μg/ml; BD Pharmingen), rabbit anti-VE-cadherin (4 μg/ml; Cayman, Cedarlane laboratories) and mouse anti-alpha-fetoprotein (1 μg/ml; abcam). After incubation with either Alexa 594- or 488- conjugated secondary antibodies the coverslips were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen, molecular probes), and visualized under fluorescence microscopy.

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized for 15 min in 0.1% NP-40 in phosphate-buffered saline (PBS). Cells were blocked using 1% of normal goat serum for 1 h at RT. The following antibodies were used: rabbit anti-OCT4 (Abcam), mouse anti-TRA-1-60 (Millipore), rabbit anti-NANOG (Stemgent), mouse anti-SSEA-4 (Millipore), rabbit anti-TH (Calbiochem), mouse anti-SOX2 (R&D Systems), mouse anti-mouse anti-Nestin (Santa Cruz), mouse anti-Pax6 (Santa Cruz). Corresponding Alexa Fluor-conjugated secondary antibodies were from Thermo Fisher. To label nuclei, coverslips were mounted using DAPI Fluoromount-G (SouthernBiotech). Images were acquired using a confocal microscope (Zeiss LSM710) operated by the ZEN software package. ImageJ (Schneider et al., 2012 (link)) and Adobe Photoshop CS6 (Adobe, United States) software were used for final image processing.

For immunofluorescent staining, we fixed cells on glass slides with a 4% paraformaldehyde solution for 10 min, washed three times with PBS, and permeabilized with PBS containing 0.1% Triton X-100 for 10 min at room temperature. After blocking in blocking buffer (PBS containing 2% bovine serum albumin) for 1 h at room temperature, the samples were incubated with primary antibody diluted in blocking buffer at 4 °C overnight. The following primary antibodies were used: rabbit anti-OCT4 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-SSEA4 (1:200, Millipore, Billerica, MA, USA), rabbit anti-NESTIN (1:1000, Millipore), goat anti-SOX17 (1:200, Santa Cruz Biotechnology), mouse anti-α-SMA (1:400, Sigma-Aldrich), mouse anti-CD31 (1:200, BD Biosciences, San Jose, CA, USA), and rabbit anti-vWF (1:500, Millipore). After washing three times with PBS, we incubated samples with fluorescence-tagged secondary antibodies (Alexa Fluor^® 488 or Alexa Fluor^® 594, 1:1000, Invitrogen) in PBS for 30 min at room temperature. Samples were washed again three times with PBS and mounted onto slides using 4′,6-diamidino-2-phenylindole-containing mounting medium (Vector Laboratories, Burlingame, CA, USA). All images were captured with a fluorescence microscope (Eclipse Ti-U, Nikon Instruments Inc., Tokyo, Japan).