Alexa fluor 564

We followed the methods described previously (Neugebauer et al., 2009 (link)) for immunostaining. The mixed immunofluorescence and in situ hybridization technique was adapted (Thisse and Thisse, 2008 (link)) as follows: on the second day, we added the antibody anti-GFP together with the antibody Anti-Digoxigenin-AP Fab Fragments (Roche) and incubated over night at 4°C in a horizontal rotator; on the third day, we added the secondary antibody anti-rabbit Alexa Fluor 488 and incubated over night at 4°C in a horizontal rotator; on the fourth day, we developed the RNA probe with Fast-Red substrate (Roche) until a red deposit was observed and the reaction was stopped with 4% PFA (in PBS) for 5 min. her12 RNA probe was a gift from Leonor Saude. Antibodies used were anti-acetylated α-tubulin (1:300; T7451 from Sigma), anti-GFP (1:500; ab290 from Abcam), anti-DlD (1:50; zdd2 monoclonal antibody [Itoh et al., 2003 (link)]), anti-mouse Alexa Fluor 564, and anti-rabbit Alexa Fluor 488 (1:500; Invitrogen). Nuclei were stained with DAPI (1:500). Flat-mounted embryos were examined with a Zeiss LSM 710 Meta confocal microscope and a Zeiss 40x water immersion C-Apochromat lens (1.2 NA). Three-colour confocal z-series images were acquired using sequential laser excitation, and analysed using Fiji software (LSM Reader) (Schindelin et al., 2012 (link)).

For immunofluorescent staining of hepatocytes, cells were seeded on cover slips and fixed in 4% paraformaldehyde for 15 min at RT, washed with PBS 3 times for 5 min each and blocked with PBS containing 5% goat serum and 0.3% Triton-X100 for 1 hr. Cells were then incubated with primary antibodies against Ki-67 (ab16667; Abcam), albumin (NB600-41532, Novus), phospho-cJun (Ser73, D47G9, 3270, Cell Signaling), cMyc (D84C12, 5605, Cell Signaling) overnight at 4°C. After washing in PBS, cells were incubated with anti-rabbit secondary antibodies conjugated with Alexa Fluor 564 (Invitrogen), Alexa Fluor 488 (Invitrogen), or FITC (Sigma), for 1 hr at RT. The cells were washed in PBS and incubated with Flash Phalloidin Red 594 antibody (424203, Biolegend) for 30 min at RT. Following three washes in PBS, cells were mounted with mounting media containing DAPI. Images were taken by fluorescence microscope (Zeiss).

Cells fixed in 4% paraformaldehyde, 4% sucrose were permeabilised in 0.1% Triton,% BSA and stained with primary antibodies (mouse anti-FLAG (M2), 1:1000; rabbit anti-GFP, 1:500) in 1% BSA in PBS for 2 h at room temperature. Secondary antibodies (anti-mouse Alexa Fluor-564 and anti-rabbit Alexa Fluor-488; Invitrogen, Paisley, UK) were applied at 1:500 in 1% BSA in PBS for 1 h in the dark. Images were acquired with a RoleraXR CCD camera (QImaging) on a Nikon TE2000 epifluorescence microscope controlled by SimplePCI software (Hamamatsu).