647 donkey anti rabbit igg

Immunostaining was performed according to the standard methods. In brief, embryos were dissected and fixed overnight with 4% PFA/PBS at 4 ˚C. After the fixation, embryos were heated in 10 mM sodium citrate buffer pH6.0 at 80 ˚C for 30 min by a block incubator (ASTEC: BI-525A, sample holder: TM-15). After cooling on ice, samples were incubated for the blocking with 10% NDS in 0.1% PBST at room temperature for 1 h. Anti-Pax6 (rabbit; Medical & Biological Laboratories #PD022) and Cdh2 (rat; DSHB, MNCD2) were used as the primary antibody at dilution of 1:1000 (Pax6) and 1:10 (Cdh2). For the secondary antibody, Alexa Fluor 488 donkey anti-rat IgG (Invitrogen, A-21208), 647 donkey anti-rabbit IgG (abcam, ab150075) were utilized at dilution of 1:600. DAPI (1:10000; Invitrogen) staining was performed to visualize nuclei. Fluorescent images were acquired under BZ-X700 (KEYENCE).

Anti-Pax6 (rabbit; Medical & Biological Laboratories #PD022), Pax7 (mouse; DSHB, PAX7-s), FoxA2 (mouse; DSHB, 4C7), β-galactosidase (β-gal: chicken; abcam, ab9361) and GFP (rabbit; MBL, 598) were used as the primary antibody at dilution of 1:1000 (Pax6), 1:10 (Pax7), 1:20 (FoxA2), 1:1000 (β-gal) and 1:1000 (GFP), respectively. For the secondary antibody, Alexa Fluor 488 donkey anti-chicken IgG (Jackson Immuno Research, 703-545-155), 647 donkey anti-mouse IgG (abcam, ab150111) and 647 donkey anti-rabbit IgG (abcam, ab150075) were utilized at dilution of 1:600. Immunostaining was performed according to the standard methods. DAPI (1:10000; Invitrogen) staining was performed to visualize nuclei. Fluorescent images were acquired under BZ-X700 (KEYENCE).