For smFISH, 2–4 h or 1–3 h embryos were processed as described (Hoppe et al., 2020 (link)) with anti-sog Stellaris (Fig. 2C, Fig. S1), or sog single molecule inexpensive FISH (smiFISH) (Fig. S4) (Tsanov et al., 2016 (link)), ush Stellaris, lacZ Stellaris, and Race smiFISH probes (Tsanov et al., 2016 (link)). ush probe sequences are available from (Hoppe et al., 2020 (link)), while sog Stellaris, sog smiFISH, lacZ Stellaris, and Race smiFISH probe sequences are provided in Table S2. Race and sog smiFISH probes were annealed to a 570-conjugated Y-FLAP and Z-FLAP, respectively (Tsanov et al., 2016 (link)). For immunostaining against mNeonGreen and Spectrin, mouse anti-mNeonGreen [32F6] (1:500, ChromoTek, cat. #32f6-100, RRID: AB_2827566), mouse anti-Spectrin (1:50, DSHB, cat. #3A9 (323 or M10-2), RRID: AB_528473) and donkey anti-mouse IgG secondary antibody, Alexa Fluor 488 (1:1000, Thermo Fisher Scientific, cat. #A-21202, RRID: AB_141607) primary and secondary antibodies were used. To stain nuclei, samples were incubated with DAPI (1:1000, NEB 4083). Samples were mounted in ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific, P36961).
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