Anti mmp2 antibody

Xenografted tumors from the mice were excised, fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin for histological studies. Paraffin-embedded tissues were sectioned into slices of 4-µm thickness for histological studies. Dewaxed and rehydrated tissue sections were subjected to antigen retrieval processes. Upon blocking, the sections were incubated overnight at 4°C with anti-cluster of differentiation (CD) 31 antibody (dilution 1:50; catalogue number JC70; Neomarkers, Fremont, CA, USA) or anti-MMP-2 antibody (dilution 1:200; catalogue number ab37150; Abcam, Cambridge, UK). Negative controls were prepared using PBS instead of the primary antibodies. Upon washing with PBS, the sections were incubated with a goat anti-mouse EnVision kit (Genentech, South San Francisco, CA, USA) for 40 min at 37°C, and then incubated with 3,3′-diaminobenzidine chromogen for 10 min. The slides for CD31-periodic acid-Schiff (PAS; Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) double staining were then exposed to a 0.5% periodic acid solution for 15 min and subsequently incubated in Schiff solution for 20 min in a dark chamber. Subsequently, the slides were washed with distilled water for 3 min and counterstained with hematoxylin. Multiplication of intensity and percentage scores was utilized to determine the staining index result.

Total proteins from the cells were extracted with Laemmli buffer, incubated at 90°C for 5 min and centrifuged at 12,000× g for 20 min. Protein concentrations were measured using the BCA method. For the Western blot assay, proteins were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gel (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA), blocked in 5% bovine serum albumin (BSA) with 0.1% Tween 20 in tris buffered saline (TBS) for 1 h at room temperature (RT) and, subsequently, incubated with the following primary antibodies: anti-vimentin antibody (Protein-tech, 1:3,000), anti-E-cadherin antibody (Abcam, 1:3,000), anti-ZO-1 antibody (Proteintech, 1:3,000), anti-MMP-2 antibody (Abcam, 1:3,000), anti-c-Myc antibody (Abcam, 1:3,000) and anti-GAPDH antibody (Proteintech, 1:3,000) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000, Thermo Fisher Scientific). The resultant bands were visualized using an enhanced chemiluminescence (ECL) detection system (ChemiDoc XRS; Bio-Rad, Hercules, CA, USA) and quantified using Quantity One software (Bio-Rad).

Formalin-fixed and paraffin-embedded tissues were cut into 4-mm thick sections and subjected to immunohistochemistry using En Vision staining, following the manufacturer’s instructions. The sections were initially blocked with 5% BSA and then incubated with specific primary antibodies, including anti-TIMP-2 antibody (1:50, Santa Cruz, CA, USA), anti-MMP-2 antibody (1:200, Abcam Inc, MA, UK), or anti-TGF-β1 antibody (1:150, Boshide Biotech, Wuhan, China). The incubation was performed overnight at 4°C. Subsequently, the sections were incubated with a secondary antibody, goat anti-rabbit immunoglobulin G (Santa Cruz, CA, USA). As a negative control for staining, some tissue sections were incubated with phosphate-buffered saline (PBS) without primary antibodies. For image analysis, the Optical Density (OD) was calculated using Image-Pro Plus 6.0 image processing software based on five randomly chosen microscopic fields.

Sorafenib (Nexavar^®) and SB-3CT (MMP-2 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA). LY294002 (PI3K inhibitor), MK-2206 (AKT inhibitor), rapamycin (mTOR inhibitor), RG7204 (RAF inhibitor), U0126 (MEK inhibitor), and PD98059 (ERK inhibitor) were obtained from Selleck Chemicals. For in vitro experiments, these reagents were dissolved in dimethyl sulfoxide (DMSO) for long-term storage and then diluted to the working concentration with DMEM. The final DMSO concentration was less than 0.1%. For in vivo experiments, SB-3CT was dissolved in 10% DMSO, and sorafenib was dissolved in a mixture of Cremophor EL/ethanol/ddH₂O (1:1:6). Primary antibodies for p/t-PI3K, p/t-AKT, p/t-mTOR, p/t-RAF, p/t-MEK, and p/t-ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-MMP-2 antibody was purchased from Abcam (Cambridge, UK). The antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibodies were obtained from CWBIO (Beijing, P.R. China).