Alex fluor 488

WT and Gata5/6 KD embryos at 72 hpf were fixed in 4% PFA at 4°C overnight. After 3 × 10 min washes in PBSTw, embryos were bleached in bleach buffer (400 μl of 100% KOH, 150 μl of 30% H₂O₂, and 50 μl of Tween 20 to a final volume of 5 ml with H₂O) in the dark until the pigment was fully eliminated. Embryos were then rinsed twice with PBSTw and permeabilized in PBS with 3% Triton (PBSTx) for 4 hours at room temperature (RT). Blocking was performed in PBS with 5% normal goat serum (Millipore, catalog no. S26-LITER) and 0.075% saponin for 1.5 to 2 hours at RT before incubation with the primary antibody [Elnb, 1:500 (72 (link))] at 4°C overnight. The next day, embryos were washed several times for 4 to 6 hours with PBSTx at RT followed by incubation of the secondary antibody (α-rabbit immunoglobulin G, Alex Fluor 488, 1:1000; Thermo Fisher Scientific, catalog no. A-11008) at 4°C overnight. After 3 × 10 min washes in PBSTw, embryos were mounted in low-melt agarose for imaging under a Nikon A1R Si point scanning confocal microscope at ×20 and ×40 magnification.

We used primary antibodies at the following concentrations: guinea pig anti-Mxc (1:5000; gift from Drs. Robert Duronio and William Marzluff), rabbit anti-GFP (1:1000; ThermoFisher Scientific #A-6455), rabbit anti-FLASH (1:2000, gift from Drs. Robert Duronio and William Marzluff), guinea pig anti-Mute (1:5000; Bulchand et al. 2010 (link)). We used secondary antibodies (ThermoFisher Scientific) at a concentration of 1:1000: goat anti-guinea pig AlexFluor 647 (#A-11073), rabbit anti-guinea pig TRITC(#PA1-28594), goat anti-rabbit AlexFluor 488 (#A-21450).

Brains were fixed overnight in 4% paraformaldehyde (PFA) at 4°C, followed by submersion in 30% sucrose until sinking, as previously described (Silver et al. 2010 (link)). Brain cryostat sections (20 µm) were prepared and stored at −80°C until use. Sections were permeabilized with 0.25% Triton X-100 for 10 min and blocked with MOM block reagent (Vector laboratories) for 1 h at room temperature (RT). Sections were incubated with primary antibodies for 2 h at RT or overnight at 4°C. Sections were then incubated in species appropriate secondary antibodies and Hoechst for 15 min at room temperature. The following primary antibodies were used: rabbit anti-CC3 (diluted 1:200; Cell Signaling); rabbit anti-TBR2 (1:1,000; Abcam); rabbit anti-PAX6 (1:1,000; Millipore); mouse anti-TUJ1 (1:400; Covance). The following secondary antibodies were used: Alex Fluor 488 and Alex Fluor 594 (1:400; Invitrogen). Hoechst (Thermo Fisher Scientific) was used for nucleus counterstain. High-magnification images were captured using a Zeiss Axio Observer Z.1 microscope coupled with an apotome. Cortical thickness was measured with Zen software. Cell quantification was performed with ImageJ/FIJI. Three sections from anatomically comparable regions per embryo and three biological replicates from control (wild-type) and mutant alleles (Casc3^RRU345/+, Casc3^{RRU345/RRU345}, and Casc3^RRU345/Null) were quantified.

hiPSC-CMs were dissociated and seeded on Matrigel coated 12mm coverslips. After one week, cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized in 5% BSA-PBS containing 0.3% triton. Cells were incubated with primary antibodies (see Key Resources Table) overnight at 4°C. After washing, cells were incubated with secondary antibodies Alex Fluor 488, 555, or 647 (Invitrogen) / Hoechst 33342, and mounted with Fluoromount-G (Southern Biotech). All images were collected on a Zeiss LSM 880 confocal system with a 40x/63x objective and using airyscan imaging mode, followed by airyscan processing using Zeiss Zen software.