Sarcomeric α actinin

Noon of the day a vaginal plug was observed was defined as 0.5 dpc. Embryonic hearts were hematoxylin and eosin stained using standard protocols. S1pr1 expression was assessed in S1pr1^+/LacZ embryonic hearts using whole mount X-gal staining followed by 4% PFA post-fixation overnight and either paraffin or cryosectioning. For antibody staining, whole embryos collected up to 13.5 dpc and excised embryonic hearts collected from 14.5 to 18.5 dpc were snap frozen and immunostained as previously described (Wilsbacher and Coughlin, 2015 ) using sarcomeric α-actinin (Sigma A7811) and fibronectin (Sigma F3648) primary antibodies. Measurements of compact and trabecular myocardial wall thickness were obtained from sections of embryonic hearts collected at 12.5, 13.5, 15.5, and 16.5 dpc from controls and mutants using Fiji image analysis software (Schindelin et al., 2012 (link)). Measurements were taken at two to six evenly spaced intervals along the left ventricular free wall of each section. For one control and one mutant heart at both 15.5 dpc and 16.5 dpc, only high power images of the compact wall were obtained; trabecular wall measurements were not made in these hearts. The average of measurements from each heart was used for statistical analysis (see Statistics).

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.