Ab32152

After conventional deaffinity and rehydration, the high-pressure antigen was extracted with sodium citrate solution at pH 6.0 for 3 min, and the sections were incubated at 30°C for 30 min. After blocking endogenous peroxidase with hydrogen peroxide for 30 min, incubated with sections of primary antibody VEGF (Abcam, ab32152, 1:250) overnight at 4°C. The next day, sections were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, ab97080, 1:200) for 30 minutes. After PBS washing, sections are stained with DAB, hematoxylin staining, dehydrated, gel fixation.

Skin tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer on ice for 30 min. We measured protein expression from the cell lysate (30~60 mg) using IL-6 (ab6672, Abcam, MA, USA), IL-10 (12163, CST, MA, USA), VEGF (ab32152, Abcam, MA, USA), MCP-1 (ab25124, Abcam, MA, USA), and β-actin (3700, CST, MA, USA). Briefly, the samples were loaded onto 15% SDS gel to separate the proteins by polyacrylamide gel electrophoresis. Then the proteins were transferred onto the PDVF membrane (ISEQ00010, Millipore, MA, USA). Primary antibodies were incubated for 12 h at 4 °C. Washed membrane and then incubated it with the HRP-conjugated antibody, and finally conducted with ECL. The gray value of the bands was analyzed using a gel analyzer (Media Cybernetics, MD, USA).

Total protein was isolated from lung tissue homogenates. The lysate was centrifuged at 11,000 g for 1 min at 4°C. Supernatants were collected and the protein content was quantitated using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). Equal amounts of total protein were separated using 12% SDS polyacrylamide gels, and then transferred on to PVDF membranes (Millipore, USA). After blocking with 5% milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C, membranes were incubated with rabbit anti-actin monoclonal antibody (1:1000 dilution, ab179467, Abcam), rabbit anti-VEGF Receptor 1 monoclonal antibody (1:2000 dilution, ab32152, Abcam), or rabbit anti-PGF polyclonal antibody (1:1000 dilution, TA332424, OriGene Technologies, USA) at 37°C for 1 h. After washing with TBST three times, all membranes were incubated with goat anti-rabbit IgG H&L (HRP) preadsorbed secondary antibody (1:5000 dilution) (Abcam) at 37°C for 40 min. After washing with TBST three times, protein expression was visualized using Immobilon Western Chemilum HRP Substrate (Millipore). Actin served as an internal loading control. Relative protein expression was calculated using a method described previously (25 (link)).

To assess the angiogenic potential of the extract, human umbilical vein endothelial cells (HUVECs) were used. A density of 5,000 HUVECs were seeded in a 48-well plate and incubated with the extract for 7 days. The medium was refreshed every other day. After incubation, the cells were stained for primary VEGF antibody (ab32152, Abcam, UK), FITC-labelled F-actin and the fluorescent secondary antibody, followed by incubation in the dark for 30 min. VEGF expression within the cells was observed under CLSM. The fluorescence intensity was semi-quantitatively analyzed using Image Pro Plus software.
Matrigel (Corning, USA) was used to prepare a substrate for cell seeding in cell culture plates. A density of 5,000 HUVECs were seeded and incubated with the extract for 12 hours. Tubular network formation was observed under an optical microscope. The spacing between the networks was semi-quantitatively measured using Image J software.

All the animal experiments were complied with the guidelines of the Tianjin Medical Experimental Animal Care, and animal protocols were approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., ltd (No. YSY-DWLL-2023227). Sprague-Dawley rats after being anaesthetized by isoflurane, the full thickness skin defect was created by 10 mm biopsy punching on the back of the rats, then the animal models could be divided into four groups. (1) No further treatment, leaving the wound alone (set as control groups). (2) The wound was covered by Tegaderm™. (3) The wound was covered by SIS. (4) The wound was covered by SIS/PAA/LAP. After implanting the materials, the implanted materials were fixed and covered by the sterile bandage to prevent the implant from dropping or separating from the wound. All the implanted materials were refreshed every two days, followed by the standard of care. At 3-, 6-, 9-day post-surgery, the gross appearances of the wound regeneration were estimated, among them, the harvested located tissues were fixed in 10% formalin and sectioned for H&E staining, Masson’s trichrome staining and VEGF/DAPI (ab32152, Abcam) immunohistochemical staining. The images were captured on the digital slice scanning equipment (Nanozoomer, Hamamatsu, Japan).

Eugenia caryophyllata Thunb (EC), Myristica fragrans Houtt (MF), Moschus berezovskii Flerov (MB), and Crocus sativus L (CS), which were identified by Dr. Lin Dong (Department of Pharmacognosy, Ningxia Medical University), were purchased from Ming De Chinese Herbal Pieces Co., Ltd. (Ningxia, China), and the specimens (#20150701, #20150703, #20150704, and #20150705) were preserved in the Department of Pharmacy, Ningxia Medical University. Nimodipine was provided by Bayer Healthcare Co., Ltd. (Germany); nitric oxide (NO, 20181221), super oxide dismutase (SOD, 20181221), malondialdehyde (MDA, 20190308), glutathione (GSH, 20181218), and glutathiol (GSSG, 20190306) reagent kits were offered by the Institute of Nanjing Jiancheng Biological Engineering (Nanjing, China); antibodies of vascular endothelial growth factor (VEGF, ab32152), caspase-3 (ab32351), and nuclear factor-κB (NF-κB, ab32536) were obtained from Abcam Co., Ltd (Shanghai, China); acetonitrile, methanol, isopropanol, and formic acid of HPLC grade were obtained from Fisher Scientific (Nepean, Canada) and Sigma-Aldrich (St. Louis, United States); ultrapure water was obtained from Watsons Water (Guangzhou, China); and triphenyltetrazolium chloride (TTC) was obtained from Suolaibao Engineering (Beijing, China). All the other reagents were of analytical grade.

We performed Western blot analysis as previously described (3). Tibial growth plate specimens were centrifuged, and the supernatants were separated by SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane (Millipore). The membranes were incubated with rabbit-source antibodies to FGF2 (Abclonal Technology, #A0235, 1:1,000), FGFR1 (Abclonal Technology, #A2073, 1:1,000), VEGFA (Abcam, #ab46154, 1:1,000) and VEGFR1 (Abcam, #ab32152, 1:1,000), and then re-probed with appropriate horseradish peroxidase-conjugated secondary antibodies (Servicebio technology, #GB23303, 1:3,000). The membranes were then visualized by enhanced chemiluminescence (ECL Kit, Servicebio technology, #G2014) and exposed to X-ray films.