Bovine serum albumin (bsa)

Cells were cultured on coverslips in 6-well plate overnight at 37 °C incubator. Then cells were fixed with 4% cold paraformaldehyde and permeabilized with 0.5% Triton X-100, blocked with 3% bovine serum albumin (Servicebio, Cat# G5001) for 30 min at room temperature. After washing with PBS for 3 times, cells were then incubated with primary antibody against PHF14 (ThermoFisher Scientific, Cat# PA5-72775, 1:200) at 4 °C overnight. Cell nuclei were stained by DAPI (Servicebio, Cat# G1012). The cytoskeleton was visualized through staining with anti-stain 488 phalloidin (Servicebio, Cat# G1028-Green) according to manufacturer’s instructions. Cy3 conjugated goat anti-rabbit IgG antibody (Servicebio, Cat# GB21303, 1:300) was used to detect the primary antibody. Finally, the slides were scanned using Pannoramic MIDI (3D HISTECH, Hungary).

Six tissue chips (TFHCC-01), each containing 90 HCC tissues and 90 para-carcinoma tissues, were purchased from TUFEIBIO (Shanghai, China). To perform IHC staining, the tissues were dewaxed and dehydrated using a mixture of ethanol and xylene. Then, they were immersed in a sodium citrate solution (0.1 M, pH 6.0; Solarbio, Shanghai, China) to retrieve antigens through high temperatures and high pressures. To prevent nonspecific binding in the tissues, a blocking solution with 0.3% hydrogen peroxide and 5% bovine serum albumin (Servicebio, Wuhan, China) was applied. Following this, primary antibodies were introduced, and the tissues were incubated overnight at 4 °C. After being washed three times with phosphate-buffered saline, the tissues were exposed to secondary antibodies conjugated with HRP (Proteintech, Wuhan, China) for a duration of 2 h. Subsequently, they were stained using HRP-diaminobenzidine. Subsequently, the presence of the antigen-antibody complex was identified in tissues through the utilization of an orthotopic light microscope. The primary antibodies used for targeted proteins were as follows: m¹A (1:100), DCAF8L1 (1:100), CDK5R2/p39 (1:50), PAGE1 (1:50), TRIM36 (1:50), and CYP26B1 (1:50).

Frozen sections or paraffin sections in groups were prepared as described above. Re-epithelialization and scar formation were assessed by H&E staining (Servicebio, China). Collagen deposition was detected by Masson’s trichrome staining (Servicebio, China). For immunohistochemical staining, the sections were blocked with 1% bovine serum albumin (Servicebio, China) and 0.5% Triton-X100 (Servicebio, China) and then separately treated with AE1/AE3 (Abcam, USA, prediluted), CD31(Abcam, USA, 1:500), CD68 (Abcam, USA, 1:500) and P63 (Abcam, USA, 1:500) mouse anti-human IgG antibody at 4 °C overnight. After washing three times with PBS for 5 min, the sections were incubated for 1 h with an HRP goat anti-mouse IgG antibody (Invitrogen, USA, 1:1000) at room temperature. Finally, the sections were stained with 3,3 N-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. The slides were covered with coverslips and examined under a microscope.

Fetal hearts on GD18.5 and adult hearts on 24 weeks were fixed with 4% paraformaldehyde (Servicebio, G1101), paraffin-embedded, processed, and sectioned at 4 μm. Hematoxylin and eosin (H&E) reagent (Servicebio, G1003), FITC-conjugated wheat germ agglutinin (WGA) (sigma, L4895), and Masson (Servicebio, G1006) were used to evaluate the cardiomyocytes cross-sectional areas and the extent of myocardial fibrosis, respectively. More than 200 randomly cardiomyocytes were selected to calculate the cross-sectional area for one slide. Fibrosis area was assessed by randomly selecting at least three fields from one slide. Cell size and fibrotic area were measured by ImageJ.
For immunohistochemistry, the slides were deparaffinized, rehydrated, and antigen retrieval. Then, the slides were immersed in 3% H₂O₂ (Servicebio, G0115) and incubated at room temperature for 20 min, followed by 3% BSA (Bovine Serum Albumin) (Servicebio, GC305010) blocking for 30 min at room temperature. The sections were incubated with Ki67 reagent (Servicebio, GB111141) at 4 °C overnight. After three washes, the sections were incubated with Goat anti-rabbit with HRP (Servicebio, G1213). Finally, the positive cells detected by 3,3′-diaminobenzidine (DAB) (Servicebio, G1212) were shown brown. The ratio of positive cells reflecting the proliferation of cardiomyocytes was measured by ImageJ.

All tissue samples were fixed in 4% paraformaldehyde solution for 12 h, and tissue samples were embedded using paraffin wax. Hematoxylin kit (Servicebio) and Eosin staining kit (Servicebio) were used for staining, followed by sealing with neutral gum (Servicebio). For Ki67 staining, closure was performed using 5% bovine serum albumin (Servicebio). The samples were then incubated with anti-Ki67 polyclonal antibody (1:800 dilution) (Servicebio) and fluorescently labeled secondary antibody (1:2000 dilution) (Zhuangzhi, Shaanxi, China), and captured by fluorescence microscopy. Immunohistochemical staining was performed using tumor tissue sections from TNBC patients. The sections were permeabilized and blocked with 3% hydrogen peroxide, and then incubated with CCDC150 primary antibody (CUSABIO, Wuhan, China). The images were captured by fluorescence microscopy.

Forty HCC samples and tumor tissues were embedded in paraffin. After dewaxing and dehydration with xylene and an ethanol gradient, the tissues were immersed in 0.1 M sodium citrate solution (pH 6.0; Sigma-Aldrich, USA) and subjected to antigen retrieval using a high-temperature and high-pressure method. To reduce nonspecific binding, a solution containing 0.3% hydrogen peroxide and 5% bovine serum albumin (Servicebio, Wuhan, China) was used to block the tissues. Primary antibodies against HIF1α (1:100; Cat No. 20960-1-AP; Proteintech, Wuhan, China), carbonic anhydrase 9 (CA9; 1:200; Cat No. 11071-1-AP; Proteintech, Wuhan, China), CFHR3 (1:100; Cat No. 16583-1-AP; Proteintech, Wuhan, China), and proliferating cell nuclear antigen (PCNA; 1:200; Cat No. 10205-2-AP; Proteintech, Wuhan, China) were incubated overnight at 4°C. Following three washes with phosphate-buffered saline (PBS), the tissues were incubated with horseradish peroxidase-conjugated secondary antibodies (Boster, Wuhan, China) for 2 h and then stained with horseradish peroxidase- diaminobenzidine reagent (Beyotime, Suzhou, China). Finally, an orthotopic light microscope was used to detect antigen-antibody complex signaling in tissues.