Rabbit anti rat laminin

After each step of incubation with antibodies, samples were washed with PBS three times for 10 min each time. Neonatal CMs cultured on aligned collagen-coated coverslips were fixed at culture stages of 4, 18, 48, and 72 h. The fixed sample was first stained for α-actinin: It was blocked with 1% BSA, 4% donkey serum, 0.025% Triton X-100, and PBS for 30 minutes at RT. Next, the sample was incubated with the first antibody, mouse-anti-rat α-actinin (1:500, Sigma, US), overnight at 4 °C. It was then incubated with the second antibody, Alexa fluor © 488 conjugated donkey-anti-mouse IgG (1:200, Life Technologies, US), for 1.5 h in the dark at RT. The sample was stained for laminin and blocked again with 1% BSA, 4% goat serum and PBS for 30 minutes at RT. It was incubated with the first antibody, rabbit-anti-rat laminin (1:200, Abcam, US) overnight at 4 °C. It was then incubated with the second antibody, Alexa fluor© 594 coagulated goat-anti-rabbit IgG (1:200, Life Technologies, US), for 1.5 h at RT in the dark. After being mounted with ProLong© Antifade Kit medium (Life Technologies, US), the sample was ready for observation.