Pascal 5 confocal microscope

The ESSCs were assayed for neural lineage commitment on different days using the Neural Stem Cell Marker Characterisation Kit (Chemicon, Millipore, USA), which included antibodies against nestin, Sox2, Map2, GFAP, and oligodendrocyte marker O1. All of the procedures were done in accordance with the manufacturer's instructions. The cell samples were further analysed by laser scanning microscopy (Pascal 5 confocal microscope, Carl Zeiss, Germany).

The BM MSC and neurosphere phenotypes were determined by immunological labelling. Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature and blocked with a solution containing 5% of bovine serum albumin in PBS and 0.05% Triton X-100 for cell permeabilization. Immunocytochemistry was carried out according to standard protocols. Cell nuclei were counterstained with SYTOX® Blue nucleic acid stain (1:1000; Invitrogen, USA). The antibodies used and dilution were as follows: Anti-CD90 (FITC) conjugated mouse monoclonal antibody (1:200; Thermo Scientific, USA); Anti-CD44 (PE) conjugated rat monoclonal antibody (1:200; Abcam, Cambridge, UK) [17 (link)]; Alexa Fluor® 647 mouse anti-nestin (1:200; BD Pharmingen, USA); Milli-Mark™ FluoroPan neuronal marker (1:100; Catalog # MAB2300X, Millipore, USA); Anti-Sox-2 clone 6G1.2 FITC conjugated antibody (1:100; Millipore, USA); Rabbit anti-GFAP (1:100; Millipore, USA) and donkey anti-rabbit IgG-FITC secondary antibody (1:250; Santa Cruz Biotechnology, Inc, USA). UltraCruz™ anti-fading mounting medium (Catalog # Sc-24941, Santa Cruz Biotechnology, Inc, USA) was applied to minimise photo bleaching due to exposure to high-intensity light. The fixed cells were then viewed using a Pascal 5 confocal microscope (Carl Zeiss, Germany).