Cfi60

The cultured cells were washed one time with cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Following washing three times with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS buffer for 20 minutes at room temperature. Following washing three times with 0.1% Tween20 in PBS, the cells were blocked with 0.1% Tween20 and 10% goat serum, with 1% BSA in PBS buffer for 1 hour at room temperature. The cells were incubated with the anti-rabbit primary antibodies of β-catenin (Abcam, Cambridge, UK), Axin1 (Novus Biologicals, Centennial, USA), Axin2 (Novus Biologicals) and phosphorylated LRP6 (Biorbyt Ltd., Cambridge, UK) over night at 4°C. The cells were washed three times with 0.1% Tween20 in PBS. The cells were incubated with Alexa Fluor^® 568 conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, USA) for 45 minutes at room temperature. To visualize the nuclei, the cells were double-stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, USA). The cells were viewed with a Keyence BZ 800 epifluorescence microscope, which was equipped with a digital camera (CFI 60, Nikon Corporation, Tokyo, Japan). All immunofluorescence images were obtained with identical exposure settings.

Ovaries were dissected from 3–4-day-old females in PBS then transferred and fixed in PBS plus Triton X-100 (PBT) (PBS with 0.1% Triton X-100) containing 0.1% glutaraldehyde for 5 min and rinsed three times with PBT. Each sample was incubated in a 0.2% X-gal staining solution (10 mM phosphate buffer, pH 7.2, 1 mM MgCl2, 5 mM K4[FeII(CN)6], 5 mM K3[Fe(III)(CN)6], 0.1% Triton X-100) for 45 min. After staining, each sample was rinsed three times with PBT, mounted in PBS containing 50% glycerol, and analyzed by bright-field microscopy on a Nikon Eclipse 90i microscope equipped with a 12V, 100W halogen lamp by using a Nikon CFI60 (Chromatic Aberration Free Infinity) Plan Fluor 20x objective with numerical aperture 0.50 and Nomarski optics. Digital images were acquired with a Nikon Digital Sight camera and assembled using the Adobe Photoshop software. No biased image manipulations were applied.

Acridine orange staining was performed to investigate cellular apoptosis in ethanol exposed embryos, following the protocol by Kim et al. (2014). Acridine orange can permeate apoptotic cells and binds to DNA, whereas healthy cells are nonpermeable to acridine orange. Thus, it stains necrotic or late apoptotic cells. Five embryos from each treatment were exposed to ethanol at 24 hpf, for 2 hr. After that, animals were washed twice, transferred to 96‐well plates, and treated with acridine orange solution (7 μg/ml) for 1 hr, at 28 ± 1°C in a dark room. Next, embryos were washed twice and anesthetized by ice before observation under the fluorescence microscope (Nikon CFI60, Eclipse Ti). Animals were photographed on the same settings to standardize the background: DSQi1Mc 12 bit, auto‐exposure 10 s, and analog gain of 16.0×. For each larva, the fluorescence intensity was quantified using the ImageJ software 1.52p (National Institutes of Health, USA).