Ssea 1

Circulating PGCs derived from HH14 embryonic blood or cultured PGCs were adhered onto a MAS-GP Type A coated
glass slide (Matsunami Glass, Osaka, Japan) and fixed with 4% paraformaldehyde for 5 min at room temperature
(RT). After several washes, cells were blocked with PBS containing 5% normal goat serum or Image-iT signal
enhancer (Life Technologies) and incubated overnight at 4ºC with primary antibodies. Then, cells were
incubated for 30 min or 1 h at RT with secondary antibodies. Subsequently, cells were counterstained with 1
µg/ml Hoechst 33342 (Dojindo, Kumamoto, Japan). Fluorescent images were captured using an Eclipse E1000
fluorescence upright microscope (Nikon, Tokyo, Japan), and these images were processed using Photoshop
Elements (Adobe Systems, San Jose, CA, USA) for trimming and overlaying. Sources and dilution of used
antibodies were as follows: rat anti-chicken vasa homolog (CVH) raised in our laboratory (1:10000) [27 (link)], mouse anti-stage specific embryonic antigen-1 (SSEA-1; 1:100,
Developmental Studies Hybridoma Bank, Iowa City, IA, USA), mouse anti-chicken c-KIT (1:500, SouthernBiotech,
Birmingham, AL, USA), goat anti-rat IgG conjugated with Alexa Fluor 488 (1:1000, Life Technologies) and goat
anti-mouse IgG or IgM conjugated with Alexa Fluor 594 (1:1000, Life Technologies).

Embryos fixed in 4% PFA were embedded in OCT and cryosectioned at a thickness of 10 µm. Slides were blocked 1 h in 10% calf serum + 0.1% Triton X-100 in PBS and stained overnight at 4°C in the blocking buffer followed by three 15-min washes in PBS. Primary antibodies used included β-catenin (rabbit, 1:25; #9582s; Cell Signaling Technology), Axin2 (goat, Conductin M-20, 1:100; #sc-8570; Santa Cruz Biotechnology, Inc.), GFP (chicken, Aves GFP-1020, 1:200), SSEA1 (mouse IgM, 1:200; MC-480; Developmental Studies Hybridoma Bank), Vasa (rabbit, 1:400; #ab13840-100; Abcam), cleaved PARP (rabbit, 1:50; #9544s; Cell Signaling Technology), E-cadherin (rat, 1:200; #13–1900; Invitrogen), Wnt5a (goat, 1:20; #AF645; R&D Systems), and Stella (rabbit, 1:100; #ab19878; Abcam). Histological staining for β-catenin was preceded by 10 min additional fixation in 4% PFA and 3 min treatment in undiluted Ficin (Invitrogen) at room temperature followed by a quick PBS wash. This treatment was not necessary for β-catenin staining on cultured cells. EdU was labeled per kit protocol (#C10338 or C10340; Thermo Fisher Scientific). Secondary antibodies were purchased from Invitrogen and incubated for 1 h in blocking buffer at room temperature at 1:200. Nuclei were labeled with DAPI or Hoechst (1:1,000; Roche or Sigma-Aldrich). Sections were mounted in Vectashield (Vector Laboratories).