Cardiac troponin t

Immunofluorescence staining of iPSCs with pluripotent markers was
performed exactly according to manufacturer instructions using the PSC
4-marker immunocytochemistry kit (ThermoFisher Scientific).
Immunofluorescence staining was performed on iN/glia co-cultures using the
following antibodies mouse anti-MAP2 (Sigma-Aldrich, 1:500, E028 rabbit
anti-synapsin (E028, 1:1000), Alexa 488- and Alexa 555-conjugated secondary
antibodies (Invitrogen, 1:5000). Briefly, cultured iN cells were fixed in
4% paraformaldehyde in PBS for 10 min, washed two times with PBS,
and permeabilized in 0.2% Triton X-100 in PBS for 5 min. Cells were
blocked in PBS containing 5% CCS, 0.5% BSA, and
0.02% NaN3 for 1 hr. Primary antibodies were applied over night at
4°C and cells were subsequently washed in PBS for three times.
Secondary antibodies were applied for 1 hr at room temperature washed in PBS
for three times. Cell nuclei were counterstained with 0.1μg/ml DAPI
(Thermo Scientific) in PBS for 10 min. Immunostaining of differentiated
iPSC-CMs was performed using cardiac troponin T (cTnT, Thermo Scientific),
sarcomeric α-actinin (Clone EA-53, Sigma), and DAPI (Thermo
Scientific) as previously described(Sun et
al., 2012). Labeled cells were examined and imaged by confocal
microscope (Carl Zeiss, LSM 510 Meta) at 20 × to 63 ×
objectives as appropriate.

MEFs were seeded and reprogrammed by GHMT or GHMT2m in 12 well plates. On days 9 and 12, cells were rinsed 1× in ice-cold PBS and fixed in 2% paraformaldehyde for 10 min at room temperature. Cells were washed 3× in PBS and permeabilized with 0.2% Triton X-100 for 15 min at room temperature. Cells were then blocked in 10% horse serum (Gemini). Primary antibodies (Cardiac Troponin T; Thermo Scientific ms-295-p - 1:400; α-actinin; Sigma A7811L - 1:400; Cardiac Troponin I; Phosphosolutions 2010-TNI – 1:400) were diluted in 10% horse serum (Gemini) and added to fixed cells for 1 h at room temperature. Cells were washed 3× in PBS and then incubated with diluted secondary antibodies (anti-mouse Alexa 555; Life Technologies A-21422 - 1:800; anti-mouse Alexa 488; Life Technologies A11034–1:800) and Hoechst (Life Technologies 62,249–1:10,000) for 1 h at room temperature in the dark. Cells were then washed 3× in PBS and imaged an EVOS FL Color Imaging System (Life Technologies).

In situ DeadEnd™ Colorimetric Apoptosis Detection System (Promega, Madison, WI, USA) was performed to detect apoptotic cells in frozen heart sections of ischemic area according to the manufacturer’s instructions. 4″, 6-diamidino-2-phenylindole (DAPI) was used to visualize nuclei. To determine the cardiomyocyte apoptosis, heart sections were double immunostained with cardiac troponin T (1:100, ThermoFisher) and TUNEL.