Gb113151

Wound newly formed skin was collected on the 14th day post-surgery and fixed overnight at 4 °C in 4% paraformaldehyde. Following this, tissue sections were prepared using standard procedures. Hematoxylin and eosin staining (HE) was performed on some sections, while Weigert iron hematoxylin and Lixin Red staining were used for Masson staining on other sections. Immunohistochemistry was conducted using the following primary antibodies: CD31 (1:500,GB113151, Servicebio), α-SMA (1:500,GB13044, Servicebio), iNOS (1:1000, No. 22226-1-AP, Proteintech), and CD206 (1:1000, No. 18704-1-AP, Proteintech).

Anti-CD80 monoclonal antibody (66,406-1-Ig, Proteintech), anti-CD163 monoclonal antibody (GB13340, Servicebio), anti-CD8 monoclonal antibody (GB12068, Servicebio), anti-CD31 monoclonal antibody (GB113151, Servicebio), VEGFA Monoclonal antibody (19,003-1-AP, Proteintech), anti-CD47 monoclonal antibody (ab218810, Abcam), carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (C0051, Beyotime), PerCP anti-CD68 (333,813, BioLegend), PE anti-CD11b (101,208, BioLegend), granulocyte–macrophage colony-stimulating factor (GM-CSF) (C003, novoprotein), FITC-labeled anti-CD47 (CC2C6, BioLegend), Human Lymphocyte separation medium (7,111,011, DAKEWE). The fusion protein SIRPα-Fc has been engineered based on the initial extracellular domain of SIRPα and is currently undergoing Phase I/II clinical trial (NCT05140811) [32 (link)]. SIRPα-VEGFR1 is constructed by combining the extracellular domain of SIRPα with that of VEGFR1 (GenBank accession number: MG920788).

After paraffin sections were rehydrated, slices were incubated in an antigen retrieval solution and blocking serum. Then, the primary antibodies, α-SMA (Servicebio, GB13044, 1:300), CD31 (Servicebio, GB113151, 1: 1,000), transforming growth factor-β₁ (TGF-β₁, Servicebio, GB13028, 1: 200), tumor necrosis factor-α (TNF-α, Servicebio, GB13452, 1: 200), interleukin-4 (IL-4, Bioss, bs-0581r, 1: 200), interferon-γ (IFN-γ, Proteintech, 15365-1-AP, 1: 2000), iNOS (Proteintech, 18985-I-AP, 1: 1,000), and CD206 (Novus, NBP1-90020, 1: 1,000), were added at 4°C overnight. Next, these sections were incubated with HRP-labeled goat anti-rabbit IgG secondary antibody for 50 min at room temperature. Subsequently, the sections were reacted with DAB solution after being washed in PBS, and the nuclei were counterstained with DAPI. Images of smear specimens were also collected by the inverted fluorescence microscope.

Immunohistochemistry has been performed as previously described using the following antibodies:
KI67 (GB111499, 1:300, Servicebio, Wuhan, China)
CD31 (GB113151, 1:500, Servicebio, Wuhan, China)
DABtunel (G1507-50, 1:5:50, Servicebio, Wuhan, China).

The slices were fixed with 10% formalin and embedded in paraffin for immunofluorescence staining. Tissue slices from tumor areas were then deparaffinized and subjected to antigen retrieval using citrate buffer three times for 10 min each in a microwave oven, followed by sequential incubation in 30% hydrogen peroxide in methanol and blocking with 0.5% BSA for 30 min. Tissue slices were then incubated with the following primary antibodies: anti-CD31 (1:300, GB113151, Servicebio) and anti-ki67 (1:300, GB121141, Servicebio). The secondary antibodies used were Alexa Fluor CY3-conjugated anti-mouse (1:300, GB21301, Servicebio) and Alexa Fluor 488-conjugated anti-rabbit (1:400, GB25303, Servicebio). A microscope was used for imaging.

After the mice were killed, we sampled multiple (> 3) longitudinal section from the central region of every tumor and performed pathological staining. Prior to analysis, the images were reviewed, and slides displaying evident non-specific staining or tissue folding were excluded. Subsequently, one histopathological section from the tumor center was subjected to analysis.
Immunostaining of endothelial cells in tumor sections was performed with a rat anti-mouse CD31 (Servicebio, GB113151) for vessel density analysis. Smooth muscle cells and pericytes were labeled using biotinylated anti-α-smooth muscle actin (αSMA, Servicebio, GB13044) for vessel maturation analysis. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Fluorescent micrographs were obtained with a Nikon Eclipse C1 microscope and DS-U3 camera control unit (Nikon, Tokyo, Japan).
Tumor vessel density (VD-H) was calculated by dividing the area of CD31-positive structures by the area of the manually selected tumor region 21 (link), 43 (link)-48 (link). We then calculated the ratio of CD31 and αSMA positive regions to only CD31-positive regions to determine the VMI (VMI-H) 46 (link), 49 (link), 50 (link).
All analyses were performed with MATLAB 2020b.