Slicing machine

Mice were anaesthetized with isoflurane and perfused with 4% paraformaldehyde prepared in PBS (0.1 M, pH=7.5). Brain and pancreas tissues were isolated, fixed in 10% neutral formalin, dehydrated in a graded series of ethanol, embedded in paraffin, and cut into 5-μm slices using a slicing machine (Leica, Germany). For immunofluorescence, slides after deparaffinization and rehydration were placed in citric acid buffer (10 mM, pH=6.5) within a decloaking chamber at 120 °C for 5 min and cooled at room temperature for 1 h. Then, slides were washed two times in PBS for 5 min, treated with 3% H₂O₂ for 30 min and blocked with 5% BSA for 30 min at 37 °C. Sections were incubated with primary antibody SYP (1:200, cell signal technology) at 4 °C overnight. After triple washing with PBS (5 min/time), sections were incubated with anti-rabbit IgG-HRP secondary antibody (1:500, cell signal technology) at 37 °C for 1 h. Sections were washed in PBS (5 min/time) and added with DAPI (200 ng/ml) for 2 min at room temperature. Pancreas sections were incubated with primary antibody anti-insulin (Abcam, ab181547) and anti-glucagon (Abcam, ab10988) overnight at 4 °C and then incubated with secondary antibody (1:500, anti-rabbit IgG-HRP) at 37 °C for 1 h. Finally, the images were captured by using a Nikon ECLIPSE Ti microscope and processed by the ImageJ software.

In this study, mice were anesthetized using isoflurane and sacrificed via normal saline perfusion. Then, liver tissues (n = 3) were collected, fixed in a 4% paraformaldehyde solution prepared in PBS buffer (0.1 M, pH = 7.5), and subsequently subjected to dehydration via an ethanol gradient series. The tissue samples were embedded in paraffin and sectioned into 5 μm slices with a slicing machine (Leica, Germany). The liver sections were subsequently stained with periodic acid–Schiff (PAS) for evaluating the level of glycogen. In order to assess lipid droplets, liver tissues (n = 3) were collected, frozen, and embedded in optimal cutting temperature compounds. The tissue samples were sectioned into 5 μm slices with a slicing machine and stained with oil red O (ORO). The Nikon ECLIPSE Ti microscope was utilized for image acquisition, and the positive staining area was measured with ImageJ software (NIH, Bethesda, MD, USA).

Fluorescence quantitative PCR instrument (PikoReal 96) and fluorescence PCR plate (SPL0960) were obtained from Thermo (Thermo, USA). Decoloring shaker (TS-1) and vortex mixer (GL-88B) were bought from Qilinbeier (Jiangsu, China). Tabletop refrigerated centrifuge (H1650R) was obtained from Xiangyi (Hunan, China). Membrane transfer apparatus, electrophoresis apparatus, and horizontal agarose electrophoresis tank were purchased from Liuyi (Beijing Liuyi Biological Technology Co Ltd, China). Magnetic stirrers (JB‐13) were purchased from INESA scientific instrument (Shanghai, China). An optical microscope was obtained from Olympus Corporation, Japan. The slicing machine was purchased from LEICA (Germany). A multifunctional enzyme marker analyzer was obtained from Heales (Shenzhen, China). SDZ‐V nerve and muscle stimulator were purchased from Hwato (Suzhou Medical Appliance Factory, China). Primer synthesis and probe modification were conducted in Sangon Biotech (Shanghai, China).

Liver, tumor, and paracancerous tissues were isolated and fixed in 4% paraformaldehyde for 24 h. The tissue samples were embedded in paraffin and sliced using a slicing machine (Leica). Hematoxylin and eosin were used to stain the slices, which were observed using a light microscope (Olympus). The experiments were repeated three times

Fixed tissue blocks were composed within the electron microscopy fixation fluid through the process of trimming, post-ES fixation, dehydration, resin infiltration, etc. Imported epoxy resin 812 was used as the embedding agent, and the tissue specimen was embedded in the resin. Next, a Slicing Machine (Leica, Wetzlar, Germany) was used to slice resin and stain with uranium acetate and lead citrate. Then the dyed copper mesh was observed on the Transmission Electron Microscope (Hitachi, Ltd., Tokyo, Japan) which captured the reasonable view.

Liver tissue was isolated and fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for 48 h before the embedding in paraffin and slicing by a slicing machine (Leica, Solms, Germany). The sections were stained with hematoxylin and eosin (HE) for pathological observation and examined under a light microscope (Olympus, Tokyo, Japan). Photographs were taken as indicated (200× magnification).

Portions of 5 mm × 5 mm × 5 mm large yellow croaker dorsal muscle were sampled and placed in Carnoy fixative [ethanol: glacial acetic acid (v/v) = 3:1] for 24 h. The samples were dehydrated stepwise with gradient ethanol series and placed into xylene to perform transparent processing. Then, the samples were embedded in paraffin to obtain 1 cm³ blocks and sectioned with a slicing machine (Leica, Germany). The slices were stained by the haematoxylin-eosin method and observed with a light microscope (Eclipse E200 biological microscope, Nikon Instruments Co. Ltd., Tokyo, Japan).