Alexa fluor conjugated secondary antibodies

Rabbit polyclonal anti-Slc22a14 antibody was raised against a keyhole limpet hemocyanin-conjugated peptide corresponding to the C-terminal region of mouse Slc22a14 (⁶¹⁵PKMDLPVQSLKAQPP⁶²⁹). Peptide synthesis, animal immunisation, and collection of sera were performed by Sigma-Aldrich Japan. The antibody was purified by affinity chromatography using an antigen peptide-conjugated column. Anti-GAPDH monoclonal antibody, anti-human septin 4 (N) antibody, and anti-phosphorylated tyrosine antibody (4G10) were purchased from WAKO Pure Chemical Industries, Immuno-Biological Laboratories (IBL), and Merck Millipore, respectively. Horseradish peroxidase-conjugated and Alexa Fluor-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology and Life Technologies, respectively.

Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X for 5 min, and blocked with 3% BSA for 1 hours. Cells were subsequently stained with appropriate primary antibodies (Abcam) and AlexaFluor conjugated secondary antibodies (Santa Cruz). Nuclei were stained with DAPI (Roche). The primary antibodies OCT4 (Santa Cruz), NANOG (Santa Cruz), SSEA-4 (Abcam) and SOX2 (Abcam) were used for pluripotency staining of undifferentiated H9 cells. The primary antibody of CD144 (Abcam) was used for staining of H9-ECs. H9-ECs were also stained with Dil-Ac-LDL (Thermo Fisher Scientific). Images were collected using an inverted confocal microscope (Nikon) and NIS-Elements AR software.

Established iPSCs were fixed with 4% paraformaldehyde prepared in phosphate-buffered saline. Following permeabilisation with 0.3% Triton X-100, iPSCs were stained with primary antibodies against OCT3/4, SSEA4, NANOG (all from Santa Cruz Biotechnology, Inc.) and TRA-1-60 (Chemicon). iPSC-CMs were then passaged onto Matrigel-coated 12 mm glass coverslips, followed by staining with antibodies against TNNT2 (Thermo Scientific and Abcam), MLC2a (Santa Cruz Biotechnology) and SA-actinin (Sigma Chemical Co). After reaction with the primary antibodies, cells were incubated with the appropriate Alexa Fluor-conjugated secondary antibodies (Santa Cruz Biotechnology or Life Technologies).
Images of the stained cells were obtained under a bright field microscope (Leica). Confocal images were taken using a 63× Plan-Apochromat oil immersion objective (Carl Zeiss) and a LSM 510 Meta confocal microscope (Carl Zeiss). Images were analysed using ZEN software (Carl Zeiss) and ImageJ software (National Institutes of Health).