Cryostar nx50

To further detect hepatic lipid distribution, Oil Red O staining was performed as previously described (Araújo et al., 2016 (link)). Briefly, the fixed liver samples (5 per group) were embedded in the presence of liquid nitrogen. Frozen cuts were made on a cryostat (Cryostar NX50, Thermo, USA), and the cuts were fixed in fixation solution for 15 min and stained with Oil Red O for 8–10 min, after which they were rinsed with distilled water. The samples were immersed in 75% ethanol and rinsed again. Then, the nuclei lightly stained with hematoxylin were observed. Images of the same magnification (200×) were collected.

Four pieces of approximately 5 mm in length were cut transversally to the muscle fiber from the center of frozen hairtail stored at each freezing temperature with a blade previously cooled to −20 °C. The entire operation was carried out in a walk-in freezer to ensure a perfect cold chain. Cryosections were obtained according to a previous method in the literature^{17 (link)} with some modifications. The frozen pieces were embedded in OCT embedding compound and submerged in dry ice until completely frozen. Each embedded sample was mounted on a chuck and placed in a CRYOSTAR NX50 (Thermo Scientific, USA) previously cooled to −20 °C. The surface of the hairtail specimen was trimmed and subsequently covered with an adhesive film (Cryofilm type 2 C(9), Section-Lab Co, Ltd., Japan) to support the frozen sections as previously described^{18 ,19 (link)}. A14 µm thick section was carefully cut, fixed in 70% ethanol for 2 min, then stained with hematoxylin and eosin for later microscopic analysis.
All of the prepared sections were observed microscopically (UPH203i, Chongqing UOP Optoelectronic Technology Co., Ltd, China) fitted with a digital camera (JFMV-M1200C, Nanjing Yifei Technology Co., Ltd, China) at a magnification of 200×.

Healthy mouse kidneys were dissected from BALB/c mice. The mice were sacrificed by an overdose pentobarbital (200 µL of 60 mg/mL) after which the kidneys were dissected and rinsed with saline to remove blood. Pig salivary glands were obtained in collaboration with the Medanex clinic (Diest, Belgium). After dissection, tissues of interest were either prepared for snapfreezing or paraffin embedding. For snapfreezing, tissues were embedded in Tissue Tek (Tissue-Tek O.C.T., Sakura Finetek Europe B.V, Alphen aan den Rijn, the Netherlands) and snapfrozen using 2-methylbutane at −40 °C. Tissues were stored at −20 °C until further processing. For paraffin embedding, tissues were fixed using 4% PFA and stored in 70% ethanol until further use. Next, tissues were dehydrated in an ethanol/xylol series and submerged in paraffin. After hardening, tissues were stored until further use. Cryosections of 20 µm thickness were sliced using a cryotome (Cryostar NX50, Thermo Fisher Scientific, Geel, Belgium) and tissue sections were mounted on Superfrost Plus microscope slides. Slides were then stored at −20 °C until further use. Paraffin sections were sliced using a microtome at 7 µm thickness and mounted on microscope slides. Sections were stored at room temperature until further use.

A piece of LT muscle was fixed in fixative (Servicebio, Beijing, China) for 24 h, successively dehydrated in 15 and 30% sucrose solutions at 4°C, and embedded in Tissue-Tek O.C.T. compound (Sakura, Japan). The tissue blocks were sectioned into 8–10 μm slices using a cryostat (CryoStar NX50, Thermo, United States) at −20°C.
Histochemical staining of myosin adenosine triphosphatase (m-ATPase) was performed as in literature (16 (link)). Briefly, muscle sections were first pre-incubated in solution (0.17 M Tris-base, 20 mM CaCl₂, pH 10.4) for 5 min, then incubated in solution (0.17 M Tris-base, 18 mM CaCl₂, 2.7 mM ATP, pH 9.4) for 30 min. Tissue sections were stained with 2% calcium chloride for 3 times with each time for 2 min, then stained with 2% cobalt nitrate for 5 min and washed, and finally stained with 1% ammonium sulfide for 30 s and washed. Tissue sections were dehydrated successively in 70, 80, 95 and 100% alcohol, diaphanized in xylene, and sealed with neutral gum.
Histological slides were scanned using a digital slide scanner (Pannoramic DESK, 3DHistech, Hungary) and muscle fibres were counted by using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., United States). The percentages of type I and type II muscle fibers were calculated.

Wheat germ agglutinin (WGA) staining was performed on 5 μm transverse cryosections of the heart. Heart cryosections were prepared using a CryoStarNX50 (Thermo Fisher Scientific, United States) and fixed in 4% paraformaldehyde solution for 30 min. Sections were washed 2 × 5 min in TBS post-fixation and then incubated with WGA staining solution (5 mg/ml, cat# W834, Thermo Fisher Scientific, United States) for 15 min at RT. Next, sections were washed and mounted with coverslip, and images were captured using a fluorescence microscope (Olympus IX71 Imaging Systems) to measured mean cardiomyocyte diameter and numbers per unit area.

Mouse tissues were stained with hematoxylin–eosin (H&E) and Oil Red O for histopathological examination. The samples were prepared by Wuhan Servicebio Technology Co., Ltd. (Suzhou, Jiangsu, China). Mouse colons and livers were placed in a 10% neutral formalin solution and fixed in paraffin. A pathology slicer (RM2016; Leica Instrument Co., Ltd., Shanghai, China) was used to slice the fixed tissues into 4 μm-thick sections. The cells were then stained with an H&E solution. Frozen liver tissues were sectioned at a thickness of 8–10 µm using a cryostat (CryoStar NX50; Thermo Scientific, Waltham, MA, USA) and stained with an Oil Red O solution. Slides were observed under a light microscope (Eclipse E100; Nikon, Tokyo, Japan). The detailed method is described in Section S2.