Ab205402

smFISH targeting nascent RNA of HES1 or BCL11A were performed using RNAscope® Multiplex Fluorescent Reagent Kit v2(ACDBio). Probes binding the intronic region of target genes were designed and synthesized by ACDBio. FISH signal was labeled with TSA Plus Cyanine 5 (Perkin Elmer). Immunocytochemistry was carried out after FISH procedure [22 ]. Antibodies targeting GFP(Abcam, ab1218), mCherry (Abcam, ab205402), GFAP (Ab4648) and SATB2 (Abcam, ab34735) were incubated overnight. Secondary antibodies including Alexa Fluor 594 Goat anti-chicken IgY secondary antibody (Thermo Fisher Scientific, A11042), Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific, A21202), Alexa Fluor 546 donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific, A10036) and Alexa Fluor 488 donkey anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, A21206) were incubated at RT for 1hr. Nuclei are stained with DAPI for 5 min before mounting with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific, P36930).

Livers and plantar skin tissues from 6-week-old Rosa26^{CAG-LSL-NuM-mCherry};Tbx3^CreERT2 mice that had been administered tamoxifen or corn oil were collected and cryoprotected in PBS containing 20% sucrose and frozen in optimal cutting temperature compound. The samples were sectioned, immunostained, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in Tris-buffered saline for 15 min at room temperature. Then, the sections were blocked with Blocking-One Histo (Nacalai Tesque) at room temperature for 1 h, incubated with primary antibodies at 4 °C overnight, washed, and incubated for 1 h with secondary antibodies. The samples were mounted using VECTASHIELD PLUS Antifade Mounting Medium with DAPI (Vector Laboratories). The primary antibodies were anti-Tbx3 (rabbit, 1:200, ab99302; Abcam), anti-mCherry (chicken, 1:1000, ab205402; Abcam), anti-HNF4a (goat, 1:500, sc-6556; Santa Cruz Biotechnology), and anti-CD49f (rat, 1:500, 313602; Biolegend). The secondary antibodies were Alexa Fluor 488-conjugated anti-rabbit, Cy3-conjugated anti-chicken, Alexa647-conjugated anti-mouse, and Alexa647-conjugated anti-rat (Jackson ImmunoResearch, West Grove, PA, USA). All images were acquired using an Olympus FV3000 confocal microscope.