Eclipse c1 fluorescence microscope

After being fixed for 24 h, the liver and ileum tissues were embedded in paraffin, cut into 4 μm thick sections and stained with hematoxylin and eosin (H&E) to evaluate the histological characteristics. For lipid accumulation determination, the frozen liver samples embedded in OCT compound were sliced and stained with Oil Red O solution. The above stained tissue sections were observed with Nikon Eclipse E100 (Nikon Corporation, Chiyoda Ward, Tokyo, Japan). For immunofluorescence assessment, the frozen ileum slices were incubated with anti-ZO-1 and anti-occludin antibodies (1:500; Servicebio, Wuhan, China) at 4 °C overnight, followed by incubation with an FITC-conjugated secondary antibody (Alexa fluor 488 goat anti-rabbit, 1:400; Servicebio, Wuhan, China) at 37 °C in dark for 50 min. Finally, the images were observed by a Nikon Eclipse C1 fluorescence microscope (Nikon Corporation, Chiyoda Ward, Tokyo, Japan) and recorded by a Nikon DS-U3 imaging system (Nikon Corporation, Chiyoda Ward, Tokyo, Japan). The positive area density of immunofluorescence was determined using Aipathwell image analysis software (Servicebio, Wuhan, China) and expressed as the ratio of positive cumulative optical density IOD value to the tissue area, which reflected the average depth of positive signal in the whole measurement area.

H9c2 cardiomyoblasts were seeded in six-well plates with glass coverslips. After incubation and treatment as described above, the cells were fixed in 4% paraformaldehyde for 10 min and blocked with 5% bovine serum albumin in PBS for 30 min at room temperature. Subsequently, the cells were incubated overnight at 4°C with a specific primary antibody against LC3 (Proteintech #14600-1-AP, China, 1:200), followed by incubation with a secondary antibody (Beyotime #A0423, China, 1:500) at room temperature for 1 h. Nuclei were stained with DAPI for 10 min. Finally, fluorescence images were captured using an Eclipse C1 fluorescence microscope (Nikon, Japan) at corresponding excitation wavelengths, and the images were processed with ImageJ software (NIH, United States).

The apoptosis level of testicular cells was detected by TUNEL (terminal deoxynucleotidyl transferase terminal dUTP nick end labeling) staining on paraffin sections, and the steps were carried out according to the instructions of the kit. Images were observed using an ECLIPSE C1 fluorescence microscope (Nikon, Tokyo, Japan).

To detect the expression level of the key protein (HBB) in lung parenchyma tissues, proteins were extracted from three Tibetan sheep at each altitude and separated on 12% SDS-PAGE gel. They were then transferred to polyvinylidene fluoride (PVDF) membranes, and these membranes were blocked and probed against HBB with rabbit polyclonal antibody (Abmart, Shanghai, China) at 4 °C overnight. Anti-β-tubulin polyclonal antibody (Abmart, Shanghai, China) was used as an internal reference. These membranes were further incubated with goat anti-rabbit IgG antibody (Abmart, Shanghai, China) for 2 h at room temperature. Protein bands were visualized using NcmECL Ultra reagents (NCM Biotech, Suzhou, China) in an X-ray room, and quantified using the AlphaEase FC software.
To detect the expression locations of HBB in lung parenchyma tissues, one paraffin section was selected from each altitude for immunofluorescence staining. Sections were blocked with 3% BSA (Servicebio, Wuhan, China) and incubated with rabbit polyclonal antibody against HBB (Abmart, Shanghai, China) at 4 °C overnight. Then, sections were incubated with goat anti-rabbit IgG antibody labeled with FITC (Abmart, Shanghai, China) for 50 min at room temperature and counterstained with DAPI. Finally, these sections were seen using an Eclipse C1 fluorescence microscope (Nikon, Tokyo, Japan) and imaged using CaseViewer software.

Morphologic changes in skin from both CTRL and CKO mice were determined by hematoxylin and eosin (HE) staining and Masson-Fontana staining. Skin tissue specimens were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Three-micrometer-thick sections were generated and then immunohistochemically stained with antibodies against UFL1 (Bethyl Laboratories, A303-456A; dilution 1:100), Endothelin-1 (Proteintech, 12191-1-AP, dilution 1:100), or Ki-67 (Abcam, ab16667, dilution 1:200). The sections were scanned using an automated slide scanner to create high-resolution digital images (KFBIO). TUNEL staining was performed with a TUNEL detection kit (Recordbio Biological Technology) according to the manufacturer’s instructions. Images were acquired with an Eclipse C1 fluorescence microscope (NIKON).