Eclipse e600 research microscope

RNAscope™ HD detection reagents -red kit (Catalog #324500, Advanced Cell Diagnostics, Newark, CA, USA), RNAscope™ probe (Catalog # 895241-S1, Advance Cell Diagnostics), RNAscope^® target retrieval reagents (Catalog #322000, Advance Cell Diagnostics) and RNAscope^® wash buffer reagents (Catalog #310091, Advance Cell Diagnostics) were hybridized on 5 μm slides of mouse cardiac tissues following the ISH assay protocol. The images were acquired using Nikon’s Eclipse E600 research microscope (Nikon, Tokyo, Japan).

After the overnight fixation in Bouin’s solution, the specimens were dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Tissue blocks were then cut into 5-µm serial sections on a rotary microtome (Leica, Wetzlar, Germany), and incubated overnight at 37 °C. Finally, the sections were stained with hematoxylin and eosin, and observed under a Nikon’s Eclipse E600 research microscope.

To obtain gonads at various gametogenic stages, the bay scallops were collected from Yantai (Shandong Province, China) every month for a year. After being transported to the laboratory, the scallops were acclimated in filtered and aerated seawater for 3 days. About 50 individuals were randomly chosen, and their ovaries and testes were dissected. Parts of them were flash-frozen in liquid nitrogen and stored at −80°C until used for RNA extraction. The rest parts were prepared for paraffin sectioning. They were fixed in 4% paraformaldehyde for 12–24 h followed by washing twice with 1 × PBS, dehydrated in a graded methanol series, and stored at −20°C. Then, the samples were transferred to ethanol, cleared with xylene, embedded in paraffin wax, and cut into 5 μm (ovary) or 3 μm (testis) sections on a rotary microtome (Leica, Wetzlar, Germany). Serial sections were tiled on glass slides, deparaffined with xylene, hydrated with gradient ethanol to water, and stained with hematoxylin. After that, the glass slides were counterstained with eosin, dehydrated with ethanol, cleared with xylene, mounted with neutral balsam, and covered with coverslips. Finally, the sections were observed under a Nikon Eclipse E600 research microscope.