P0081

Histological and immunohistochemical analyses were conducted after gross evaluation [32 (link), 33 (link)]. Samples were fixed in 4 % paraformaldehyde for 7 days, decalcified in 10 % EDTA for 3 weeks, embedded in paraffin, and cut perpendicularly into 5 μm sections. The sections were then stained with hematoxylin and eosin (H&E) and Masson stains to estimate the cartilaginous matrix distribution. For immunohistochemical analysis, heat-induced epitope retrieval was performed in citrate buffer (P0081, Beyotime) and immunolabeled with primary antibodies (Bsm-33,409 M, Bioss) at 4 °C overnight, followed by incubation with a secondary antibody (Bs-0377R-HRP, Bioss) to detect immunoactivity. Isotype-matched negative control antibodies were used under identical conditions. Finally, all sections were analyzed under a microscope.

After deparaffinizing by xylene and hydration using graded alcohol, antigen repair was conducted using citrate antigen retrieval solution (P0081, Beyotime, Shanghai, China) at 100 °C for 15 min. Sections were treated with H₂O₂ for 30 min, then blocked for 1 h (β-catenin) or 2 h (Runx2). Diluted primary antibodies (β-catenin:1/100, ab32572 and Runx2:1/400, ab76956, Abcam, Cambridge, UK) were used to incubate the sections at 4 °C overnight. After washing with phosphate-buffered saline (PBS) five times (3 min each time), the tissue slices were incubated with secondary antibody (1:200) for 2 h and horseradish peroxidase for 30 min at room temperature. Proteins were visualized by the DAB solution kit (Vectorlabs, CA, USA).

The methodology here refers to the published literature [30 (link)]. Tissues including the thrombus and surrounding vein walls were removed after sacrifice and fixed in 4% formaldehyde, dehydrated through a concentration gradient of ethanol, transparent in xylene, and embedded in paraffin. Thin tissue sections (5 μm thick) harvested on day 2 post-modeling underwent stain with hematoxylin-eosin (HE) and were observed under a light microscope. For immunohistochemical staining, after antigen retrieval by microwave heating in citrate buffer (Beyotime, P0081), sections were blocked with 5% bovine serum albumin (BioFroxx, 4240GR005) before anti-CD41 (Abcam, ab134131) incubation at 4°C overnight. Subsequently, the goat anti-rat IgG secondary antibody (ZGBBT, ZB-2301) was applied for 1 hour at room temperature (RT) followed by the use of diaminobenzidine substrate to visualize the stain sections. The presence of brown spots in the image showed positive staining. Antibodies against CD62P (Proteintech, 60332) and CD41 were employed for immunofluorescence double-staining. The slides were incubated with Dylight 488-conjugated goat anti-rat IgG secondary antibodies (Abbkine, A23220) or Dylight 594-conjugated goat anti-mouse IgG secondary antibodies (Abbkine, A23410). 5 to 8 randomly unrepeated field areas were selected to view under ×400 magnification in an optical microscope.

Tumor tissues were fixed in 4% paraformaldehyde, and dehydrated with gradient ethanol. Then, tissues were embedded into paraffin (YA0011, Solarbio) and cut into sections with a thickness of 5 μm. The restoration was executed with sodium citrate buffer (pH 6.0, P0081, Beyotime) at 94 °C for 15 min. Subsequently, sections were sealed with 1% bovine serum albumin (BSA, ST2249, Beyotime) for one hour, and hatched with primary antibodies targeting Ki-67 (1:200, ab15580, Abcam), FOXM1 (1:250, ab207298, Abcam) and STMN1 (1:2000, ab52630, Abcam) overnight at 4 °C. The secondary antibody HRP labeled anti-rabbit IgG antibody (ab288151, Abcam) was used to incubate with sections at 37 °C for 30 min. The sections were re-stained with hematoxylin (G1080, Solarbio), and pictured under a light microscope (Olympus).

Paraffin sections of xenografts were cut into 5- μm-thick sections. Then, the sections were infused in xylene (10 min per time for three times), followed by infusion in 90% ethanol (3 min), 75% ethanol (3 min), 50% ethanol (3 min) and ddH₂O (3 min). For immunohistochemistry, the sections were boiled in antigen restoration solution (P0081; Beyotime) for 3 min and cooled naturally. Cooled sections were incubated with 3% H₂O₂ at RT for 10 min and blotted with 3% horse serum for 1 h. Next, the sections were incubated with primary antibodies at 4 °C overnight. The following steps were in accordance to the protocols provided by the manufacturer of GTVision III Detection System/Mo&Rb (GK500705; Gene Tech Company Limited, Shanghai, China). The nucleus was stained with hematoxylin. TUNEL staining was performed according to the instructions provided by the manufacturer of DeadEnd Fluorometric TUNEL System (TB235; Promega, Madison, WI, USA). The nucleus was stained with DAPI (Beyotime) for 1 min and the percentage of TUNEL-positive cells was calculated with a mercury lamp (Olympus U-RFL-T).