Bx53f inverted microscope

The samples were first immobilized in 4% paraformaldehyde (Solarbio, Beijing, China) for 24 h, then immersed in 70%, 80%, 90%, 95%, and 100% ethanol solutions for 30 min to dehydrate the samples. They were then placed in xylene for 2 h to make the samples transparent and embedded in paraffin wax for 3 h. The embedded samples were sectioned into 5 μm pieces and immersed in xylene for 20 min to dewax the samples. The sections were then immersed in a series of ethanol solutions from high to low concentrations and finally in distilled water. The sections were stained with a hematoxylin solution (Beyotime, Haimen, China) for 4 min, fractionated in hydrochloric acid and ethanol for 3 s each, rinsed in running water for 1 h, immersed in distilled water for 10 min, and dehydrated in 70% and 90% ethanol solutions for 10 min each, followed by staining with the eosin staining solution for 3 min. The stained sections were dehydrated by immersion in an ethanol solution and then immersed in xylene to make the sections transparent, and they were finally sealed and stained with gum. The sections were sealed with resin, observed under a BX53F inverted microscope (Olympus, Tokyo, Japan), and photographed.

Frozen samples were sectioned to 8 μm, fixed in 10% formalin (Solarbio, Beijing, China) for 10 min, and washed. The sections were immersed in 60% isopropanol (Sinopharm, Beijing, China) for 2 min. The sections were stained with an Oil Red O solution (Sangon Biotech, Shanghai, China) for 15 min while protected from the light. The sections were again immersed in 60% isopropanol for 5 s to remove the staining solution and washed again in ice-cold distilled water. The nuclei were stained with Mayer’s hematoxylin (Sangon Biotech, Shanghai, China) for 5 min, washed, dried, and embedded in glycerol gelatin (Xilong Scientific, Shantou, China). The sections were observed under a BX53F inverted microscope (Olympus, Tokyo, Japan) and photographed.