Mouse anti α actinin antibody

Neonatal cardiomyocytes were exposed to synthetic CPP-Ts or to the CPP-Ts sub peptide^14–39 marked with Alexa Fluor 555 Microscale Protein Labeling Kit (555/565 nm; Invitrogen A30007, MA, USA). Confocal immunofluorescence was performed as previously described^{68 (link)}, using mouse anti-α-actinin antibody 1:150 (Sigma-Aldrich A7811, MO, USA), goat anti-mouse Alexa Fluor 488 1:500 (Invitrogen R37120, MA, USA), and TO-PRO-3 probe 1:800 (Invitrogen T3605, MA, USA) for nuclear staining. For the sub peptide^14–39 internalization assay in various cell lines, only TO-PRO-3 probe was used for nuclear staining.
Images were collected in a Zeiss Axiovert (Zeiss, CA, USA) confocal microscope. Three lasers were utilized, as follows: excitation at 488 nm and emission at 505–550 nm for Alexa 488; excitation at 568 nm and emission at 585–615 nm for Alexa 555; and excitation at 633 nm and emission at 650 nm for TO-PRO-3.

IPSC-CMs were dissociated with 0.25% trypsin-EDTA and then fixed with 4% paraformaldehyde. The cells were stained with the following primary antibodies: mouse anti-α-actinin antibody (Sigma-Aldrich) and rabbit anti-troponin I antibody (Abcam), and then visualized by the following secondary antibodies: Alexa Fluor 488 donkey anti-mouse IgG (Invitrogen) and Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen). The nucleus of the cells were stained with 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI) and then observed with a confocal laser scanning microscopy FV1200 (Olympus).