Anti dig fab

In situ RNA hybridization was performed as previously described [65 (link)]. After dissection, postnatal or adult brains were fixed in 4% PFA at 4°C for 48 or 24 h, respectively, cryoprotected in 30% sucrose at 4°C overnight, cryosectioned at 20 μm on consecutive slides, and stored at −80°C. Sections were acetylated with 0.1 M triethanolamine and 2.5 μL/mL acetanhydride for 10 min and additionally fixed in 4% PFA at RT for 15 min before prehybridization for 4 h followed by 1 to 2 μg/mL probe hybridization for 16 h at 65°C. The antisense E2-2 [NM_013685], E2A [NM_011548], and HEB [NM_011544] probes were derived from plasmid constructs, whereas linearized templates were transcribed using a T7 RNA polymerase (Thermo Fisher Scientific, Waltham, MA). Anti-DIG FAB (1:2000 (Roche)) immunostaining was applied at 4°C overnight and subsequently developed in buffer containing BCIP (175 μg/mL) and NBT (100 μg/mL) for 12-48 h.

The protocol for whole-mount in situ hybridization (WMISH) to detect spatial gene expression has been described previously [89 (link)]. Briefly, sea urchin embryos were fixed in 4% paraformaldehyde solution. The fixed embryos were incubated in hybridization buffer [50% (vol/vol) formamide, 5× SSC, 1× Denhardt’s, 1 mg/mL yeast tRNA, 50 ng/mL heparin, and 0.1% Tween-20] with a concentration from 1 to 2 ng/μL digoxygenin RNA probe(s) at 60 °C for 18 h. Two post-hybridization washes were performed with hybridization buffer without RNA probe, 2× SSCT (2× SSC, 0.1% Tween-20), 0.2× SSCT, and 0.1× SSCT, each 20 min at 60 °C. Subsequently, 3 washes were performed with MABT buffer (0.1 M maleic acid, 0.15 M NaCl, and 0.1% Tween-20). Antibody incubations were performed at room temperature with 1:2000 diluted anti-DIG Fab (Roche). The embryos were washed with MABT buffer and with AP buffer (100 mM Tris·Cl (pH 9.5), 100 mM NaCl, 50 mM MgCl2, and 1 mM levamisole). 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium were used for staining. Fluorescent in situ hybridization protocol was performed as described in [90 (link)].