Bright field microscope photograph system

The embryonic specimens were fixed overnight in 4% paraformaldehyde/phosphate-buffered saline (PBS), dehydrated in ethanol, and embedded in paraffin. Sections of 5 μm were cut from wax-embedded embryos and floated onto slides coated with 3-triethoxysilylpropylamine (440140, Sigma). The slides were dried at 37 °C overnight, dewaxed through xylene, and then rehydrated through decreasing concentrations of ethanol. Next, they were stained with hematoxylin, counterstained with eosin, dehydrated, and equilibrated with xylene. Sections were photographed under bright-field microscope photograph system (Leica Microsystems, Buffalo Grove, IL, USA).

Tissues were fixed in 4% PFA overnight, washed with PBS, dehydrated in a gradient series of alcohol solutions, and embedded in paraffin. These paraffin-embedded tissues were then sectioned (5 μm) before being deparaffinized and rehydrated. Tissue sections were stained with hematoxylin, incubated in bluing solution, counterstained with eosin, dehydrated, and equilibrated with xylene. Glass coverslips were mounted with Permount Mounting Media (SP15-100; Thermo Fisher Scientific). Sections were photographed under bright-field microscope photograph system (Leica Microsystems).

Hematoxylin and eosin staining was performed as previously described
[42 (link)]. Briefly, sections were dewaxed, rehydrated, stained with hematoxylin, incubated in bluing solution, counterstained with eosin, dehydrated, and equilibrated with xylene. Glass coverslips were mounted with Permount Mounting Media (SP15-100; Fisher Scientific, Pittsburgh, PA, USA). Sections were photographed under bright-field microscope photograph system (Leica Microsystems, Buffalo Grove, IL, USA).