Alexa conjugated anti mouse or anti rabbit igg secondary antibodies

Undirected embryoid bodies (EB) were formed by placing 10,000 iPSCs in multiple wells of a 96-well non-tissue culture treated V-bottom plate (Evergreen) containing HUESM plus 10 μM ROCKi (Stemgent), and underwent brief centrifugation. After 14 d of culturing EBs were transferred into a 6 well low attachment plates (Corning) and cultured for an additional 16 days. Once harvested EBs were fixed in 4% paraformaldehyde for 20 min at room temperature and processed in 15% and 30% sucrose solutions for one day each at 4°C. EBs were then embedded in O.C.T. (Sakura Finetek) and cryosectioned. The sections were blocked in PBS containing 0.1% Triton X-100 and 10% donkey serum for 1 hr at room temperature, followed by an overnight incubation at 4°C with antibodies identifying the 3 germ layers: SMA (1:500, DAKO), AFP (1:500, DAKO) TuJ1 (1:500, Covance), Sox17 (1:500, R&D Systems). Alexa-conjugated anti-mouse or anti-rabbit IgG secondary antibodies were used (Invitrogen) along with Hoechst 33342 counterstain. Sections were set with Vectashield Hard Set Mounting Media (Vector Laboratories).

For pluripotency staining, cultures were fixed using 4% paraformaldehyde (PFA, Santa Cruz) for 12 min at room temperature. After multiple PBS washes the cells were treated with PBS containing 0.1% Triton X-100 (Sigma) and 10% normal donkey serum (Jackson Immuno Research) for 1 hr. Cells were then treated with primary antibodies including Tra-160 (1:200, Millipore), SSEA-4 (1:500, Abcam), Tra-181 (1:200, Millipore), OCT-4 (1:500, Stemgent), Nanog (1:100, Cell Signaling), and SOX-2 (1:500, Stemgent). Alexa-conjugated anti-mouse or anti-rabbit IgG secondary antibodies were used (Invitrogen). AP staining was performed with the Vector Red Alkaline Phosphatase Substrate Kit (Vector Laboratories) per the manufacturers instructions. Nuclei were counterstained with Hoechst 33342 (Sigma).

MEFs were manually removed and iPSCs were brought to single cells suspension using Accutase (Life Technologies) and plated into a 48-well plate coated with polyornithine (100 μg/mL, Sigma Aldrich)/laminin (3 μg/mL, Invitrogen) at 25,000 cells per well, in mTeSR1 (Stem Cell Technologies) and 10 μM ROCKi. Cells were allowed to recover for 2 days before being switched to custom mTeSR1 (missing five growth factors, Stem Cell Technologies) containing 10 μM SB421542 (Stemgent) and 250 nM LDN193189 (Stemgent). Media was changed completely every 2 days. At Day 11, media was gradually switched with half feeds for the first two changes to neuronal differentiation medium (Neurobasal/B27 without retinoic acid/Glutamax (2 mM)/Penicillin-streptomycin (100 U/mL-0.1 mg/mL).
At days 14 and 21, wells were washed with PBS and fixed using 4% PFA for 12 min at room temperature. After three PBS washes, cells were blocked with 0.1% Triton X-100 and 10% normal donkey serum for 1 hr. Cells were treated with primary antibodies including PAX6 (1:300, Covance), Tuj1 (1:500, Covance), and Sox1 (1:400, Stemgent). Alexa-conjugated anti-mouse or anti-rabbit IgG secondary antibodies were utilized (Invitrogen).