Bs 0230r

Analyses of embryos and explants were analyzed using immunohistochemistry according to standard whole-mount or cryostat sectioning techniques [33 (link)]. In this study, antibodies used were: anti-TUJ1 (1:1000 dilution; ab 78078, Abcam, Cambridge, UK); anti-FGF3 (produced by ABclonal, Wuhan, China; 1:200); anti-GAD65/67 (1:1000 dilution; ab11070, Abcam, Cambridge, UK); anti-parvalbumin (1:200 dilution; PV, bs-1299R, Bioss, Beijing, China); anti-FGFR1 (1:200 dilution; bs-0230R, Bioss, Beijing, China); and anti-green fluorescent protein (GFP) mouse monoclonal (ab1218, Abcam, Cambridge, Uk). Secondary antibodies were conjugated to Alexa 647 or Alexa 488 (1:1000 dilution; Jackson, Lancaster, PA, USA); FGFR1 antibody blocking peptide (bs-0230P, Bioss; 50 ng/μL); and FGF3 antibody blocking peptide (bs-1255P, Beijing, China, Bioss; 50 ng/μL).

The xenograft tumors were sectioned at 5 μm, fixed with 4% formalin (Sigma-Aldrich) and embedded with paraffin (Sigma-Aldrich). Next, the slides were incubated with anti-FGFR1 (bs-0230R; Bioss) overnight and secondary antibody (bs-0293G-HRP) for 1 h. After that, the signal was developed with DAB solution and counterstained with hematoxylin (Sigma-Aldrich) as previously described (Wang et al., 2019b (link)).

After being extracted from tissues and cells with RIPA buffer (Beyotime), the proteins (20 μg) were separated through 10% SDS-PAGE electrophoresis. Then the proteins were transferred onto PVDF membranes, followed by blockage in 5% skim milk for 2 h. Subsequently, the membranes were maintained overnight with primary antibodies at 4°C and corresponding secondary antibody (bs-0293G-HRP; 1:10,000; Bioss, Beijing, China) for 2 h at room temperature. The protein blots were subjected to ECL kit (Beyotime) for visualization. The signal intensity of the proteins was determined by ImageJ software. The antibodies included multidrug resistance protein 1 (MRP1; bs-24241R; 1:2000; Bioss), p-glycoprotein (MDR1; bs-0563R; 1:2000; Bioss), B-cell lymphoma-2 (Bcl-2; bs-20351R; 1:2000; Bioss), BCL2-Associated X (Bax; bs-28034R; 1:2000; Bioss), FGFR1 (bs-0230R; 1:2000; Bioss) and β-actin (bs-0061R; 1:10,000; Bioss).