C0107

The immunohistochemistry process began with deparaffinization of the tissue sections in xylene, followed by rehydration using graded alcohol. Next, the sections were treated with 3% H₂O₂ (PV-6001, ZSGB-BIO) for 5 minutes and subsequently blocked for 30 minutes using 5% goat serum (16210064, Gibco). After this blocking step, the liver tissues were incubated overnight with specific antibodies against TXNIP (Huabio, ET1705-72, 1:200) and c-caspase-3 (Asp175) (Cell Signaling Technology, 9661, 1:200). Following three PBS washes, the sections were further incubated with an enzyme-conjugated secondary antibody (PV-6001, ZSGB-BIO) for 60 minutes. Staining was completed using DAB chromogen (ZLI-9017, ZSGB-BIO), and the nuclei were counterstained with hematoxylin (C0107, Beyotime) for a brief period of 3 seconds. The resulting images for every sample (200× magnification) were scanned with a pathological section scanner (HS6, SUNNY INSTRUMENT CO., LTD). The specific antibodies utilized for staining are detailed in Table S2.

Rat adenohypophysis tissues were fixed in 4% paraformaldehyde (MA0192, Meilunbio, Dalian, China) for 48 h and then made into paraffin sections. After dewaxing and rehydration, the sections were antigenically repaired using EDTA antigen retrieval solution (P0084, Beyotime, Shanghai, China). The samples were sequentially incubated in blocking solution (incubated for 30 min at room temperature), primary antibody (FTO antibody 1:2000 dilution, FOXP2 antibody 1:200 dilution, overnight at 4 °C), and secondary antibody (1:500 dilution, incubated for 1 h at room temperature away from light). The antibodies used were as follows: FTO antibody (ab280081, Abcam, UK); FOXP2 antibody (20,529–1-AP, Proteintech, USA); and anti-rabbit IgG (H + L) antibody (5220–0336, SeraCare, USA). Both antibody incubations were followed by washing three times with PBS (5 min at room temperature). The nucleus was restained with hematoxylin staining solution (C0107, Beyotime, Shanghai, China) after development using a DAB horseradish peroxidase color development kit (P0203, Beyotime, Shanghai, China). The sections were dehydrated and sealed. Images were visualized and collected using an Olympus fluorescence microscope. Three randomly selected images from the immunohistochemistry results were analyzed for positive reaction areas using ImageJ for statistical analysis.

Tissues including heart, liver, spleen, lung, kidney and tumor were dissected, embedded in paraffin, sectioned, and stained with H&E using routine methods. Briefly, tissues were resected and fixed overnight in 4% paraformaldehyde solution (Sangon Biotech, China) at room temperature. Subsequently, fixed specimens were embedded in paraffin, divided into 5 μm-thick sections, and then stained with hematoxylin staining solution (C0107, Beyotime) and eosin staining solution (C0109, Beyotime). The prepared slides were photographed by a microscope (IX51, Olympus).

After antigen retrieval in water bath and normal goat serum (C0265, Beyotime) blocking, the slides were incubated with primary antibodies to Caveolin-1 (ab32577, 1:2000, Abcam) or collagen I (ab270993, 1:500, Abcam) (Fang et al. 2018 (link)), which was followed by incubation with secondary antibody goat anti-rabbit IgG (ab6721, 1:1000, Abcam). Finally, the staining processes were performed with diaminobenzidine colorimetric reagent solution (P0203, Beyotime) and hematoxylin (C0107, Beyotime). HE staining was used for histological observation as previously described (Liu et al. 2021 (link)).

Aortic root was snap-froze in optimal cutting temperature compound, and 4 um frozen sections were cut and the endogenous peroxidase activity blocked with Hematoxylin staining solution (P1000A, Beyotime Biotechnology) for 10 min. Antigen retrieval was performed with quick antigen retrieval solution (P0090, Beyotime Biotechnology), then sections were blocked with 5% normal serum, and incubated with the primary antibody (1:100, ab125212, Abcam) for 24 h at 4 °C, followed by biotinylated secondary antibody (P0101, Beyotime Biotechnology) diluted 1:100, and counterstain with hematoxylin (C0107, Beyotime Biotechnology) for 1 min. Clear the tissue slides in 3 times of xylene and coverslip using mounting solution, then sections were observed under microscopy.