Kit 9706

Immunohistochemistry was used to examine the expression of N-cadherin in xerografted tumors. The harvested tumors were fixed in 4% paraformaldehyde for 24h. Four-micrometre thick sections were prepared from the paraffin-embedded blocks and antigenic retrieval was performed in citric acid buffer (pH 6.0). After cooling at room temperature, sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity. After incubation with blocking buffer (goat serum, MaiXin, China, KIT-9706) at room temperature for 20 minutes, sections were incubated with primary antibody N-cadherin (CST, Rabbit mAb, #13116), Vimentin (CST, Rabbit mAb,#5741), and p-Smad2 (Ser465+Ser467)/p-Smad3 (Ser423+Ser425) (Wanleibio, Rabbit, pAb, WL02305) at 4°C overnight. Then sections were incubated with second antibody (MaiXin, China, KIT-9706). After incubation with horseradish peroxidase-labeled streptavidin, antibody binding was visualized using 3,3′-diaminobenzidine (DAB), and the sections were counterstained with hematoxylin. The negative controls were sections stained without the primary antibody. Sections were evaluated and photographed at 400× magnification under microscope.

Paraffin-embedded tissue sections (4 μm) were subjected to antigen retrieval, incubation with anti-CD31 (ab28364, abcam), anti-VEGF-A (BA0407, Boster), anti-HIF-1A (PB0245, Boster), anti-CD34 (BA0532, Boster), or anti-TSP1 (ab93653, abcam) antibodies, followed by incubation with a peroxidase-conjugated goat anti-rabbit IgG (KIT-9706, Maixin-Bio) secondary antibody. Samples were developed with DAB or AEC and counterstained with Mayor's hematoxylin (AR0005, Boster). Image Pro Plus 6.0 software was used for quantitative analysis.

Tissues were harvested and fixed with 10% formalin and embedded in paraffin. Serial sections (4 μm) were prepared and stained with H&E and immunohistochemistry (IHC). For IHC, tissue sections were subjected to antigen retrieval and incubation with primary antibody against CCL2 (BF0556, Affinity), TGF-β (AF1027, Affinity), IL-1β (AF5103, Affinity), IL-6 (DF6087, Affinity), or IL-10 (DF6894, Affinity), followed by incubation with a peroxidase-conjugated goat anti-rabbit IgG (KIT-9706, Maixin-Bio) secondary antibody, and the immunoreactivity was detected with an UltraSensitive™ Streptavidin-Peroxidase Kit (KIT-9710, Mai Xin, China) according to the manufacturer's protocol. The collagen content was assessed by Sirius Red (365548, Sigma). For immunofluorescence (IF), cryosections (4 μm) were prepared from freshly frozen liver tissues and incubated with antibodies specific for mouse CD68 (137005, Biolegend), CD31-antibody (102432, Biolegend), TIE2 (124008, Biolegend), or phos-NF-κB (Affinity, AF5006). Photomicrographs were produced using a confocal microscope and corresponding software (Zeiss). Image Pro Plus 6.0 software was used for quantitative analysis.