After blocking and permeabilization, cryosections were incubated with primary antibodies against VEGFR-2 (rabbit, polyclonal, 1:100, ab39256; Abcam) and placed in a fridge (4°C) overnight. After washing with PBS, secondary FITC-coupled anti-rabbit IgG (goat, 1:400, F6005; Sigma Aldrich) or Alexa Fluor 488-coupled anti-rabbit IgG (goat, 1:400, A-11008, Molecular Probes) antibodies were added overnight at 4°C. Nuclear counterstaining was done with bisBenzimide Hoechst 33342 trihydrochloride (1:1000, B2261; Sigma-Aldrich). Further steps of immunohistochemistry were done in the same manner as just described (group 1 + 2) to label PCs.
Ab39256
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab39256 is a laboratory equipment product offered by Abcam. It is a core function device designed for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab28364, by Abcam (3 mentions)
Ab46154, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab2350, by Abcam (2 mentions)
Orbitrap elite mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Sequencing grade modified trypsin, by Promega (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Ecl reagent, by Keygen Biotech (2 mentions)
Ab9485, by Abcam (2 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
F6005, by Merck Group (1 mentions)
Bisbenzimide hoechst 33342 trihydrochloride, by Merck Group (1 mentions)
B2261, by Merck Group (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ab22614, by Abcam (1 mentions)
Ab7280, by Abcam (1 mentions)
Ab25404, by Abcam (1 mentions)
Ab52627, by Abcam (1 mentions)
Anti ephb4, by Santa Cruz Biotechnology (1 mentions)
17 protocols using ab39256
1
Immunohistochemical Localization of VEGFR-2
2
Shear Stress-Induced Protein Expression
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Cells grown in glass slides were treated with shear stress for 2 h. Then total protein was obtained by using M-PER protein extraction buffer, and quantified using a BCA kit (Thermo Fisher Scientific, Inc.) and separated on 7.5 or 12% polyacrylamide gel followed by transfer onto an ImmunBlot PVDF membrane (GE Healthcare Life Sciences, Little Chalfont, UK). The membranes were blocked for 1 h with 5% BSA in Tris-phosphate buffer containing 0.05% Tween-20 (TBS-T). It was further incubated overnight at 4°C with anti-Notch1 (1:2,000; ab52627), anti-DLL4 (1:1,000; ab7280), anti-Hey1 (2 μg/ml, ab22614), anti-Hey2 (2 μg/ml; ab25404) all from Abcam (MA, USA); anti-EphrinB2 (1:1,000; sc-398735) and anti-EphB4 (1:1,000; sc-130081) both from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-VEGFR2 (1:2,000; ab39256) and anti-CD31 (1:500; ab28364) both from Abcam, primary antibodies. After three washes (5 min) with TBS-T, membranes were further incubated with HRP-conjugated secondary antibodies: anti-rabbit (cat. no. 7074) or anti-mouse (cat. no. 7076) both from Cell Signaling Technology, Inc., (Danvers, MA, USA) for 1-2 h and followed by three washes with TBS-T. The target protein signal was detected and digitized using ECL (Thermo Fisher Scientific, Inc.).
3
Ovarian VEGF Immunohistochemical Analysis
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Immunohistochemical analysis was performed according to a well-established method ( 20 (link) ). Frozen ovarian tissue sections were air-dried for 30 min at room temperature. These sections were washed with PBS twice for 5 min each. The sections were treated with 3% hydrogen peroxidase in methanol to quench endogenous peroxidase activity and subsequently washed with PBS. Ovarian tissue sections were incubated with a blocking solution for 7 min at room temperature in a humidified chamber. These sections were incubated with VEGF (Abcam, ab46154, dilution; 1/50), VEGFR1 (Abcam, ab2350, dilution; 1/25) and VEGFR2 (Abcam, ab39256, dilution; 1/50) antibodies at +4°C overnight. The following day, the sections were incubated with SignalStain Boost IHC Detection Reagent (Cell Signaling, 8114). The reaction products were visualized using Dab (Cell Signaling, #8059). These sections were counterstained with Mayer’s haematoxylin and mounted with Entellan. Then, images were taken with a light microscope.
4
VEGFR2 Protein Interactome Profiling
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Myc-DDK (Flag)–tagged VEGFR2 (CAT#: RC219851; Origene, Rockville, MD) was stably overexpressed in RF24-par cells. These cells were then treated with VEGF (10 ng/ml) + Bev (5μg/μl), and the nuclear extracts were prepared according to the manufacturer’s instructions (Thermo Fisher Scientific). These nuclear extracts were incubated with anti-Flag M2 affinity gel (Sigma-Aldrich) overnight at 4°C, and the pulled-down complex was pH-adjusted by adding 50 mM ammonium bicarbonate and digested by adding 200 ng modified sequencing-grade trypsin (Promega, Madison, WI) for 18 hours at 37°C. The resulting peptides and compound were analyzed by LC-MS/MS on an Orbitrap-Elite mass spectrometer (Thermo Fisher Scientific) as described previously (Chan et al., 2013 (link)). The expression of VEGFR2 on the pulled-down complex was validated by applying an anti-VEGFR2 antibody (ab39256; Abcam, Cambridge, MA).
5
Western Blot Analysis of Signaling Proteins
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The HepG2, HepG2‐LR, Huh7 and Huh7‐LR cells were lysed in RIPA buffer. Protein concentration of each sample was detected by using BCA protein assay kit (Beyotime). Then, proteins were separated by 10% SDS‐PAGE gels and transferred to a PVDF membrane. The membrane was incubated with the indicated primary antibodies at 4°C over‐night. After incubated with HRP‐linked secondary antibody, the immunoreactive bands were detected with a chemiluminescence kit (Thermo Fisher Scientific). The primary antibodies include anti‐VEGF Receptor 2 antibody (1:1000, ab39256; Abcam), anti‐RAS antibody (1:1000, ab52939; Abcam), MEK1/2 (47E6) Rabbit antibody (1:1000, 9126; CST), Phospho‐MEK1/2 (Ser217/221) (41G9) Rabbit antibody (1:1000, 9154; CST), ERK1/2 (137F5) Rabbit antibody (1:1000, 4695; CST), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit antibody (1:1000, 4370; CST), anti‐GAPDH antibody (1:1000, ab8245; Abcam) and ETS‐1 (D8O8A) Rabbit antibody (1:1000, 14069; CST).
6
Protein Expression Analysis in Tumor Tissues
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Protein lysates were used to extract the proteins in the tumor tissues. Protein concentrations were determined by the bicinchoninic acid (BCA) colorimetric protein assay method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to resolve the proteins, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Merck, Darmstadt, Germany) at 80 V for 30 min. Membranes were blocked with 5% nonfat dried milk powder in Tris-buffered saline (TBS) and Tween 20 (TBST) solution for 1 hr. The primary antibodies included a rabbit polyclonal anti-COX-2 antibody (1: 1000) (ab102005, Abcam) an anti-Ang1 antibody (1: 1000) (Ab183701, Abcam), an anti-Ang2 antibody (1: 500) (ab8452, Abcam), an anti-HIF-1α antibody (1: 1000) (ab82832, Abcam), and anti-VEGF antibody (1: 2000) (ab39256, Abcam), which were incubated on the membranes overnight at 4°C. The membranes were incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 2000) (Proteintech, Chicago, Ill, USA) for 1 h. The enhanced chemiluminescence (ECL) system was used to detect the signals on the membranes. The protein expression level was standardized with GAPDH, and quantification of band intensity was performed by Image J software (National Institutes of Health, USA).
7
Western Blot Analysis of Angiogenic Signaling
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Total protein extracted from cells lysis buffer supplemented with protein inhibitor, phosphatase inhibitor and PMSF (KeyGEN BioCHEM, Nanjing, China.) and an equal amount of 30 µg protein was separated by 10% SDS-PAGE gel and transferred to polyvinylidence difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% bovine serum albumin (BSA, USA) in TBS-tween, followed by incubating with primary antibodies: anti-VEGFA(ab52917, Abcam, UK), anti-HMGA2 (#8179, Cell Signaling Technogy, USA), anti-VEGFR2 (1:1000, ab39256, abcam, UK), anti-phospho(Y1214)-VEGFR2 (1:1000, ab5475, abcam, UK), ant-Akt (#9272, cell signaling technology, USA), anti-phospho(Ser473)-Akt (1:2000, #4060, cell signaling technology, USA), ant-mTOR (1:1000, #2983, Cell Signaling Technogy, USA), anti-phospho(Ser2448)-mTOR (1:1000, #5536, Cell Signaling Technogy, USA), rabbit anti-N-cadherin (1:1000, 22018-1-AP, proteintech, China), rabbit anti-E-cadherin (1:1000, 20874-1-AP, proteintech, China), rabbit anti-vimentin (1:1000,10366-1-AP, proteintech, China) and rabbit anti-GAPDH (1:10,000, ab9485, abcam, UK). Proteins were then detected by enhanced chemiluminescence system (ECL) reagent (KeyGEN BioTECH, China) after incubation with secondary antibodies for 1 h at room temperature.
8
Ovarian Tissue Immunofluorescence Analysis
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As previously described, immunofluorescence analyses were performed ( 19 (link) ). Frozen ovarian tissue sections were air-dried for 30 min at room temperature. These sections were washed with PBS twice for 5 min each and incubated with 2.5% normal goat serum (Vector, S-1012) for 1h at room temperature in a humidified chamber. Subsequently, these sections were incubated overnight at +4°C with VEGF (Abcam, ab46154, dilution; 1/100), VEGFR1 (Abcam, ab2350, dilution; 1/100) and VEGFR2 (Abcam, ab39256, dilution; 1/500) antibodies. The next day, the sections were incubated with secondary antibodies for 45 min in darkness.
9
Quantifying Wound Healing Biomarkers
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Tissues from the amputation wound and dorsal cutaneous and tail wounds were collected on days 7, 14, and two months after the surgery and aerosol GCB treatment. Tissue sections were formalin fixed and paraffin embedded. Decalcification was performed for the amputation wound.
Mast cell numbers were quantified using toluidine blue staining and counting the number of toluidine blue positive cells in 10 high-power field (h.p.f) under a 20x objective lens.
The total number of macrophages was quantified using anti-F4/80 antibody staining (ab6640, Abcam USA). M1 macrophages were identified and quantified using iNOS staining (PA5-16855, Thermo Fisher Scientific). Anti-mannose receptor antibody (ab 64,693, Abcam USA) was used to identify and quantify M2 macrophages. CD31 antibody (ab 28,364, Abcam USA) was used for endothelial cell staining; anti-VEGFR-2 (ab39256, Abcam USA) was used for VEGF staining; IL-10 and FGF-2 were measured using ab189392 (Abcam USA) and sc-1360 (Santa Cruz Biotechnology), respectively. Ki67 antibody (ab15580, Abcam USA) was used for cell proliferation and fibroblast activation; protein alpha antibody (ab53066, Abcam USA) was used for fibroblast staining. All the sections were incubated with the primary antibody overnight at 4°C in accordance with the protocols provided by companies.
Mast cell numbers were quantified using toluidine blue staining and counting the number of toluidine blue positive cells in 10 high-power field (h.p.f) under a 20x objective lens.
The total number of macrophages was quantified using anti-F4/80 antibody staining (ab6640, Abcam USA). M1 macrophages were identified and quantified using iNOS staining (PA5-16855, Thermo Fisher Scientific). Anti-mannose receptor antibody (ab 64,693, Abcam USA) was used to identify and quantify M2 macrophages. CD31 antibody (ab 28,364, Abcam USA) was used for endothelial cell staining; anti-VEGFR-2 (ab39256, Abcam USA) was used for VEGF staining; IL-10 and FGF-2 were measured using ab189392 (Abcam USA) and sc-1360 (Santa Cruz Biotechnology), respectively. Ki67 antibody (ab15580, Abcam USA) was used for cell proliferation and fibroblast activation; protein alpha antibody (ab53066, Abcam USA) was used for fibroblast staining. All the sections were incubated with the primary antibody overnight at 4°C in accordance with the protocols provided by companies.
10
Tumor Vasculature Examination by IHC
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The vasculature within the tumors was examined via immunohistochemistry techniques specific for angiogenesis-mediated markers (CD31, CD34, CD44, and VEGFR2). Tissues were fixed in 10% formalin prior to paraffin embedding. Sections (4 mm thick) were deparaffinized using xylene and rehydrated in a series of ethanol solutions at concentrations ranging from 95% to 100%. Antigen retrieval was achieved by incubating samples with citrate buffer (pH 6) at room temperature, followed by heating them in microwave for 10 minutes. Endogenous peroxidase activity was blocked using a peroxidase-blocking reagent (Dako Denmark A/S, Glostrup, Denmark) for 5 minutes in a wet chamber. The sections were incubated with CD31 (Abcam ab28364, 1:100), CD34 (Abcam ab81289, 1:100), CD44 (Abcam ab51037, 1:100), and VEGFR2 (Abcam ab39256, 1:100) in EnVision™ Flex antibody diluent (Dako). Detection of antibody binding was performed via incubation with the EnVision Flex/HRP (Dako) for 30 minutes. Slides were reacted with a diaminobenzidine (DAB) solution for 2–3 minutes (EnVision Flex DAB + chromogen). The intensity and distribution of CD31-, CD34-, CD44-, and VEGFR2-specific immunostaining were assessed using the Olympus DP72 camera following hematoxylin counterstaining.
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