Anti fgf 2

Mouse brains were embedded in Tissue-Tek O.C.T. (Sakura Finetek, Alphen aan den Rijn, The Netherlands) and snap frozen in isopentane (Sigma-Aldrich) cooled on dry ice. Snap-frozen tissue was sectioned at 6 μm thickness on a cryostat (Leica CM3050S, Leica Microsystems, Wetzlar, Germany). Subsequent washes were done with TBS-Tween20 (Sigma-Aldrich) wash buffer, 3×3 min, all steps were performed at room temperature. The primary antibodies used were: anti-ANGPT2 (1:100, Abcam), anti-Msi1 (1:500, Abcam), anti-NG2 (1:100, Abcam), anti-Sox2 (1:100, Abcam), anti-VEGF (1:20, Abcam), anti-Vimentin (1:100, Abcam), anti-PDGFRα (1:100, Cell Signaling Technology, Beverly, MA, USA), anti-GFAP (1:500, Dako, Glostrup, Denmark), anti-Tubulin β3 (1:100, Millipore), anti-HuNu (1:100, Millipore), anti-FGF2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IDH1 (Dianova, Hamburg, Germany) and anti-POU3F2 (SC-2895, Santa Cruz). The secondary antibodies used were: FITC-conjugated goat anti-rabbit (1:200, Southern Biotech), FITC-conjugated goat anti-mouse (1:200, Southern Biotech), TXRD-conjugated goat anti-mouse (1:100, Southern Biotech).

The endometrial tissue was mixed with lysis buffer and the protease inhibitor. Then, the samples were homogenized and centrifuged (12,000 rpm × 10 min), and the supernatants were collected. After determining the protein concentration, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with the primary antibody overnight at 4 °C. The antibodies included anti-estrogen receptor α (ERα, Santa Cruz Biotechnology, USA), anti-progesterone receptor A (PRA, Proteintech, China), anti-hypoxia inducible factor 1α (HIF1α, R&D, USA), anti-VEGFA (Proteintech, China), anti-angiopoietin 2 (ANGPT2, Abcam, USA), anti-cyclooxygenase 2 (COX2, CST, USA), anti-prostaglandin E receptor 2 (EP2 receptor, Abcam, USA), anti-matrix metallopeptidase 2 (MMP2, Abcam, USA), anti-MMP9 (Abclone, China), anti-tissue inhibitor of metalloproteinase 2 (TIMP2, R&D, USA), anti-FGF2 (Santa Cruz Biotechnology, USA), and anti-β-actin (Proteintech, China). Next, the PVDF membranes were incubated with the fluorescent secondary antibodies (CST, USA) at room temperature for 1 h. Lastly, the protein bands were scanned using the Odyssey Infrared Imaging System (Li-Cor Biosciences, USA).

For immunohistochemistry analysis, the bone tissue (femur and tibiae) were harvested from PBS- and 5-FU-treated mice on 3 days and 6 days and processed for immunohistochemistry [22 (link)]. Tibiae were fixed in 4% paraformaldehyde (PFA) and paraffin-embedded and cut into 6-μm sections. After dewaxing and rehydrating, endogenous peroxidase was quenched for 15 min with 3% H₂O₂ in methanol. Heat-mediated antigen retrieval and enzymatic techniques were performed according to recommendations for the specific antibodies. A blocking step was performed using 10% normal goat serum and 1% bovine serum albumin (BSA) in PBS. After endogenous peroxidase and nonspecific protein block, primary antibodies were incubated overnight at 4 °C as follows: anti–FGF2 (Santa Cruz, CA, USA) primary antibodies. Polyclonal secondary antibodies were incubated, followed by incubation in streptavidin horseradish peroxidase (Invitrogen, CA, USA). Staining was developed with DAB according to manufacturer’s instructions (Invitrogen, CA, USA) and briefly counterstained in methy green before coverslipping in cytoseal permanent mounting media. Localization of positive staining was analyzed by light microscopy (Olympus CX41, Japan).