Eclipse ti confocal microscopy

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.