Donkey anti goat alexa fluor568

The primary antibodies in this study were as follows: goat anti-ephrin-B1 (R&D Systems; AF473); mouse anti-α sarcomeric actinin (Sigma-Aldrich, Cat# A7732, RRID:AB_2221571 or Abcam, Cat# ab68167, RRID:AB_11157538); mouse anti-connexin 43 (Millipore; MAB3067); rabbit anti-N-cadherin (Epitomics; 2019-1); rabbit anti-desmoplakin 1/2 (ARP American Research Products, Cat# 03-61003, RRID:AB_1541118); rabbit anti-claudin-5 (Acris Antibodies, Cat# DP157, RRID:AB_978124); mouse anti-GAPDH (Abcam, Cat# ab9484, RRID:AB_307274), mouse anti-Ryanodine Receptor (Abcam, Cat# ab2868, RRID:AB_2183051), and mouse anti-caveolin-3 (BD Biosciences, Cat# 610420, RRID:AB_397800). The secondary fluorescent antibodies used in this study were as follows: donkey anti-goat Alexa Fluor488 (Molecular Probes, Cat# A-11055, RRID:AB_2534102); donkey anti-mouse Alexa Fluor488 (Cat# A-21202, RRID:AB_141607), goat anti-rabbit Oregon-Green488 (Cat# 011038), donkey anti-goat Alexa Fluor568 (Cat# A-11057, RRID:AB_2534104), and donkey anti-rat Alexa Fluor568 (Cat# A-11007, RRID:AB_10561522), all obtained from Thermo Fisher Scientific. Cell nuclei were stained with 4′,6-Diamidino-2-phenylindole dihydrochloride-DAPI (Sigma-Aldrich, Cat# 32670) or TO-PRO–3 Iodide (642/661) (Invitrogen; Cat# T3605).

Immunocytochemistry was performed on day 10–11 after plating the cells on the PET 5 textile samples. The samples were fixed with 4% paraformaldehyde, blocked with 10% normal donkey serum (Biowest, Nuaille, France) solution, and stained with goat anti-cardiac troponin T (1:1,000, Abcam) and mouse anti-MyBPC3 (1:400, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4 °C overnight. Donkey anti-goat Alexa Fluor 568 and donkey anti-mouse Alexa Fluor 488 (1:800, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were used as secondary antibodies. The cell nuclei were stained using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, California, USA). Fluorescence was visualized with a Nikon A1R+ Laser Scanning Confocal Microscope (Nikon, Tokyo, Japan) using a Nikon Apo 60× 1.40NA oil objective and with Zeiss Axio Imager.M2 with ApoTome.2 and AxioCamHRm3 camera using a Zeiss EC Plan-Neofluar 40× 1.30NA oil objective.