V3000 inverted confocal microscope

Embryos were first permeabilised overnight in PBS+1% DMSO+1% Triton. They were then blocked in PBS+0.1% Triton+0.1% BSA+5% NGS and incubated overnight in primary antibodies as follows: rabbit anti-laminin (Sigma, L9393) at 1:50, rabbit anti-PhH3 (Abcam, ab5176) at 1:500 and mouse anti-acetylated tubulin (T 6793, Sigma) at 1:250.
The primary wash was performed in PBS+0.1% Triton+0.1% BSA, before a secondary block in PBS+0.1% Triton+0.1% BSA+5% NGS, and overnight incubation in goat anti-rabbit (Abcam, ab150083) and/or goat anti-mouse (Invitrogen, 84540) secondary antibodies at 1:250. Staining with DAPI at 1:500 and rhodamine phalloidin at 1:250 was performed with the secondary incubation. Embryos were washed thoroughly with PBS+0.1% Triton and mounted for confocal imaging on glass-bottomed dishes in 80% glycerol. All imaging was performed on an Olympus V3000 inverted confocal microscope at 30× optical magnification.
For EdU labelling, EdU was applied to live embryos in seawater at a final concentration of 20 µM for 2 h prior to fixation. Fluorescent detection of incorporated EdU was performed following the manufacturer's instructions using a Click-it EdU Alexa Fluor 647 Imaging Kit (Invitrogen) prior to primary antibody incubation.

Rehydrated embryos were permeabilised overnight in PBS + 1% DMSO + 1% Triton and incubated in a bleaching solution of 3% H2O2 + 3% formamide in 0.2X SSC. Embryos were then blocked in PBS + 0.1% Triton + 0.1% BSA + 5% NGS for 3 h. The blocking solution was then replaced, including primary antibodies as follows: rabbit anti-laminin (Sigma, L9393) at 1:50, rabbit anti-PhH3 (Abcam, ab5176) at 1:500, mouse anti-acetylated tubulin (Sigma, T6793) at 1:250. Primary antibody incubation was performed overnight at 4°C, followed by washes in PBS + 0.1% Triton + 0.1% BSA and then by a secondary block of PBS + 0.1% Triton + 0.1% BSA + 5% NGS for 3 h. Finally, this was replenished, to also include goat anti-rabbit and/or goat anti-mouse secondary antibodies at 1:250 and DAPI at 1:500 for overnight incubations. Rhodamine phalloidin (Thermofisher, R415) staining was performed overnight during secondary antibody incubation at 1:200 dilution. Embryos were washed thoroughly with PBS + 0.1% Triton. Imaging was performed on an Olympus V3000 inverted confocal microscope at 30X optical magnification.

Hybridization chain reaction (HCR) version 3 was performed on embryos as described in Andrews et al. (2020) (link). Briefly, embryos were rehydrated in NPBS + 0.1% Triton X, incubated for 30 min in bleaching solution and permeabilised in 1% DMSO, 1% Triton for 3 h. They were incubated in Hybridisation Buffer (HB, Molecular Instruments) for 2 h and then probes were added in HB overnight at 37°C. The following day probe excess was removed with Wash Buffer (Molecular Instrument). Embryos were washed in 5X-SSC + 0.1% Triton X, incubated in Amplification Buffer (AB, Molecular Instruments) for 30 min and then left overnight in the dark at room temperature in AB + 0.03 μM of each hairpin (Molecular Instruments). The next day embryos were washed in the dark in 5X-SSC + 0.1% Triton X and incubated overnight with 1 μg/mL DAPI in NPBT, then washed in NPBT and transferred in a glass-bottomed dish in 100% glycerol. Imaging was performed on an Olympus V3000 inverted confocal microscope.
Probes were generated using the following sequences from the B. lanceolatum transcriptome (Marlétaz et al., 2018 (link)): Six3/6 (BL08388), SerT (BL96109), VGlut (BL22589), Otp (BL13404), FoxD (BL10518).