Alexa fluor goat anti rabbit secondary antibody

Embryos were fixed with 4% paraformaldehyde, dehydrated in cold methanol and stored at −20 °C. For antibody staining, embryos were rehydrated in PBST (PBS with 0.1% Tween-20) for 10 min and permeabilized in PBSTX (PBST with 0.1% Triton X-100) for 30 min. Afterwards, embryos were blocked in 3% BSA in PBST for at least 1 h followed by incubation in the primary antibody, rabbit anti-phosphorylated Smad1/5/9 (1:200; Cell Signaling, 9511S) or BODIPY FL phallacidin (1:50; Invitrogen, B607) in 3% BSA in PBST at 4 °C overnight. It is noteworthy that for phallacidin staining, embryos were from batches without methanol treatment. Alexa Fluor goat anti-rabbit secondary antibody (1:400; Invitrogen, A-11037) was used to visualize signals. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (1:1,000; Dojindo, 340–07971) and cytoplasmic membranes were labelled with CellMask Deep Red (1:2,000; Invitrogen, C10046). Embryos were imaged using a Zeiss LMS 780 inverted confocal system.

For Western blot 20–30 μg of denaturized protein was loaded onto a Novex 4–20% Tris‐Glysine gradient gel (Invitrogen) and transferred to nitrocellulose filters (Schleicher & Schuell BioScience, Dassel, Germany). The membranes were blocked in Odyssey Blocking Buffer – TBS (1:1) (LI‐COR Biosciences, Lincoln, NE) and incubated with anti‐Reg3γ polyclonal antibody of Reg3γ (1:500 in Odyssey Blocking Buffer – TBS) overnight at +4°C. Antibody against rat Reg3γ was produced in rabbits by GenScript Corporation (Piscataway, NJ). After 1‐h incubation with Alexa Fluor goat anti‐rabbit secondary antibody (Invitrogen) at a 1:5000 dilution, the protein amount was detected using Odyssey Infrared Detection (LI‐COR Biosciences). The bands were quantified with Quantity One software (Bio‐Rad Laboratories).