Haematoxylin

As mentioned above, the expression of RUNX2 and p21 in aortic tissue was examined by immunohistochemistry [45 (link)]. In brief, arterial tissue sections were incubated at 65 °C for 2 h, dewaxed in turpentine twice for 10 min each; and rehydrated in 99%, 85% and 75% ethanol for 5 min each. Antigen retrieval was performed in a trypsin-EDTA solution. Next, sections were blocked with 5% BSA for 30 min at room temperature and incubated with specific primary antibodies, including anti-RUNX2 (bs-1134R, Bioss, 1:300) and anti-21 (10355-1-AP, Proteintech, 1:400) at 4 °C overnight. The following day, sections were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (PV-9000, ZSGBBIO, Beijing, China) at room temperature for 30 min. For control experiments, the primary antibody was replaced by PBS. Finally, the sections were incubated with DAB chromogenic solution (DA1015; Solarbio) for 1 min at room temperature. Nuclei were counterstained with haematoxylin (Solarbio) for 1 min at room temperature. The stained tissue was observed under a CX31 light microscope (Olympus Corporation, Japan). Images were taken at 100× magnification and images analysed using Image-Pro Plus software (version 6.0).

Tumour was obtained from mice, fixed in 4% paraformaldehyde, dehydrated in ethanol, permeated with xylene, embedded in paraffin and then cut into 5‐μm‐thick section. Section was put into antigen repair solution, heated for 10 min and cooled to room temperature. To eliminate the endogenous peroxidase, the section was incubated with 3% H₂O₂ for 15 min. After being blocked with 3% BSA, the section was incubated with proliferating cell nuclear antigen (PCNA) antibody (Affinity) overnight at 4°C and then with HRP‐labelled goat anti‐rabbit IgG secondary antibody (Thermo Scientific) for 60 min at 37°C. After colouration with 3,3′‐diaminobenzidine (DAB; MXB Biotechnologies, Fuzhou, China) and redyeing with haematoxylin (Solarbio), successively, section was observed under a microscope BX53 (OLYMPUS).

Lung tissues from six animals per group were respectively subjected to H&E staining. After fixed in 4% paraformaldehyde, the lung tissues were embedded in paraffin and cut into 5 μm sections. Haematoxylin (Beijing Solarbio Science & Technology Co., Ltd, China) and eosin (Sangon biotech, China) staining was used to visualize lung tissue lesions. For the quantification of vascular medial wall thickness, two sections of lung tissue from each animal were used for visualization, then two fields were taken from each section. We measured a 50–150 μm long artery in one field of view. A microscope (BX53, Olympus Corporation, Japan) was used for observation and photographs were taken using a camera system (DP73, Olympus Corporation, Japan).

Liver was dissected, fixed in cold 4% paraformaldehyde (PFA) overnight at 4 °C, dehydrated by an alcohol gradient (from 50% to 100%), rendered transparent in xylol, and embedded in paraffin. Then, tissue was cut on a microtome to slices of 5 µm in thickness. After deparaffinisation and rehydration, slices were stained with haematoxylin (Solarbio, Beijing, China), dehydrated by an alcohol gradient (from 50% to 95%), stained with eosin (Solarbio, Beijing, China), further dehydrated by 100% alcohol, rendered transparent in xylol, embedded in neutral resin, and finally covered by a cover slip. Samples were imaged by a biomicroscope (Leica, Wetzlar, Germany).

The porcine preadipocyte cells in six-well plates, which had been induced by differentiation medium, the preadipocyte cells were washed with PBS three times. The cells were covered with 1 mL of 4% formaldehyde per well at room temperature. After fixation for 20 minutes, PBS washed the six-well plates three times. The original oil red O solution (Solarbio, China) was added with 3:2 water to make oil red staining solution. The cells were covered with oil red staining solution for 20 minutes at room temperature and washed with PBS. The six-well plates were stained with haematoxylin (Solarbio, China) for 1 min, washed with ice water, and then observed using an OPTIKA XDS-3 microscope (Italy).
The porcine preadipocyte cells in six-well plates, which had been induced by differentiation medium, the cells were treated with 0.25% trypsin until separation, centrifuged for 3 min at 1000 rpm, and then TG content in cell homogenate was determined according to the instructions of TG assay kit (Nanjing Jiancheng Bioengineering Institute, China).

Tissues were fixed, paraffin-embedded and sectioned (5-μm continuous sections). H&E staining and Masson staining were performed. Paraffin sections were deparaffinized and rehydrated. The sections were heated in sodium citrate buffer in a microwave for antigen retrieval, washed with phosphate-buffered saline (PBS) for 5 min (three times) at room temperature, incubated in 3% H₂O₂ at room temperature for 25 min, washed with PBS for 5 min (three times) and blocked and incubated in goat serum for 30 min. The primary antibody was added dropwise (anti-FOXF2 antibody, 1:50, Abcam, Ab194427), and the sections were incubated at 4 °C overnight. The next day, the primary antibody was discarded, and the sections were washed with PBS for 5 min (three times). Then, 50 min after the addition of secondary antibody (1:200, SignalStain® Boost IHC Detection Reagent, CST, USA), the sections were washed with PBS for 5 min (three times). Finally, freshly prepared diaminobenzidine (DAB) was added for colour development, and the sections were counterstained with haematoxylin (Solarbio, China), dehydrated and mounted with neutral gum. All slides were observed and photographed under a microscope (× 200 or × 400) by a blinded investigator.