C2 laser scanning head

The cross-sectional area of cardiomyocytes was assessed using wheat germ agglutinin (WGA) staining. Cryosections were rinsed in PBS and then incubated with WGA conjugated with Alexa Fluor 488 (1:100, Invitrogen). Slides were imaged by Eclipse Ti confocal microscopy with a C2 laser-scanning head (Nikon). ImageJ software (National Institutes of Health) was used to quantify the size of each cell. The area of the digested cardiomyocytes was quantified using ImageJ software based on phase contrast images.

Retinas were collected as previously described (Crist et al., 2017 (link)). Briefly, neonates were sacrificed by isoflurane overexposure and eyes were removed and fixed with 4% paraformaldehyde (PFA, Thermo Scientific) in phosphate buffered saline (PBS, Fisher Scientific) for 1h at 4°C, then washed with PBS. Retinas were dissected and washed with PBS, permeabilized with 1% Triton-X100 (Fisher Scientific) in PBS for 30 min at RT, then blocked with CAS-Block (Life Technologies) for 30 min at RT. Retinas were incubated with primary antibodies (Supplementary Table S1) or IB4 conjugated with Alexa Fluor 488 (ThermoFisher, 1:100) overnight in 1% Triton-X100 in PBS at 4°C on a rocker. Retinas were washed with PBS, then incubated with secondary antibodies (Supplementary Table S1) for 4 hs at RT on a nutator mixer. Retinas were washed with PBS, then leaflets were cut into the retinas for flat mounting on slides under a coverslip with Fluoromount-G (SouthernBiotech). Images were taken using Eclipse Ti Confocal microscopy with a C2 laser-scanning head (Nikon). Images showing the superficial, intermediate, and deep layers were stacked using ImageJ (Schneider et al., 2012 (link)). Depth coded images were prepared using the “Temporal-Color Coder”, provided by ImageJ. ENDOMUCIN and COUP-TFII mean fluorescence images were quantified using ImageJ.

For immunostaining, hearts were fixed in 4% PFA at 4 °C overnight, embedded in OCT compound (Sakura), and sectioned at 8 μm thickness. For PCM1 staining, we used fresh-frozen (non-fixed) samples and sections were fixed in 10% formalin for 10 min. Sections were blocked with 1% bovine serum albumin (BSA), incubated with primary antibodies against PH3 (rabbit polyclonal, 1:200; Millipore), PCM1 (1:1000; Sigma), cardiac α-actinin (1:200; Sigma), cardiac Troponin T (1:200; Thermo), YAP (1:100; Cell Signaling), and smooth muscle α-actin conjugated with AlexaFluor594 (1:200; Sigma), and were further incubated with Alexa Fluor-conjugated secondary antibodies against mouse or rabbit IgG and with DAPI. For EdU staining, postnatal mice were administered an intraperitoneal (IP) injection of EdU (5 μg/g of mouse body weight) at P5, P6, P12, and P13, and we collected the hearts at P14. EdU incorporation was assessed using Click-IT EdU system (Invitrogen). Fluorescent images were captured using Eclipse Ti confocal microscopy with a C2 laser-scanning head (Nikon).