Maxillae were dissected and fixed in 4% PFA overnight. Samples were decalcified in 19% EDTA until soft enough to cut (~7 days). Processed samples were then dehydrated with 30% sucrose followed by embedding in OCT on dry ice with ethanol. Cryosections were fixed by 4% PFA. Sections were then subject to permeabilisation by 0.2% Triton X-100 (Sigma, X100), heat-induced antigen retrieval, and blocking with 3% BSA. Sections were stained by the following antibodies: anti-RFP (Abcam, Ab62341), anti-CD34 (Abcam, Ab81289 and Ab8158), anti-CD31 (Abcam, Ab7388 and Ab24590), anti-GFP (Abcam, Ab13970), anti-Arg1 (Abcam, Ab92274), anti-Met (Abcam, Ab51067), anti-HGF (Abcam, Ab83760), and anti-Ki67 (Abcam, Ab16667). Secondary antibodies included Alexa Fluor 488 (Invitrogen, A11039), Alexa Fluor 568 (Invitrogen, A11077), Alexa Fluor 633 (Invitrogen, A21052), and Alexa Fluor 488 (Invitrogen, A11008). Tyramide signal amplification (NEL744001KT, PerkinElmer) was performed for weak signals. Hoechst 33342 (Invitrogen 62249, 1:500) was used for DNA staining. Slides were mounted using Citifluor AF1 (EMS, 171024-AF1) and cover-slipped for microscopy. Zeiss Apotome or Leica TCS SP5 systems was used for acquiring images. ImageJ and Adobe Photoshop were used for image processing.
Ab51067
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab51067 is a primary antibody that can be used for the detection of Connexin 43 in various applications. The product is suitable for use in western blotting and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab52894, by Abcam (4 mentions)
Ab68141, by Abcam (4 mentions)
Ab16667, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab205718, by Abcam (2 mentions)
Ab39688, by Abcam (2 mentions)
Ab155549, by Abcam (2 mentions)
Ab199396, by Abcam (2 mentions)
Ab131442, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Ab188224, by Abcam (2 mentions)
Ab47405, by Abcam (2 mentions)
Ab32570, by Abcam (2 mentions)
Ab203491, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Ab182651, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
31 protocols using ab51067
1
Immunohistochemistry of Maxillary Samples
2
Comprehensive Profiling of RTK Signaling Pathways
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The FISH analyses were used to examine 18 genes relating the RTK signaling pathways (EGFR, ERBB2, MET, FGFR2, VEGFA, KRAS, and BRAF), cell cycle (CCND1, CCNE1, and cMYC), the RHOA pathway (RHOA and ARHGAP26/6), and genes known to have rearrangements (CLDN18, ALK, RET, ROS1, and NTRK‐1). Split‐FISH assays were carried out on FFPE specimens sliced to 4‐μm thickness, using bacterial artificial chromosome clone‐derived DNA probes. Cases positive for the split‐FISH assay were subjected to the fusion FISH assay to identify partner genes. Gene amplification was examined by FISH and IHC. A 5‐fold increase or more in FISH signals was tested for gene amplifications. Antibodies used in IHC included the Ventana I‐view PATHWAY anti‐human epidermal growth factor receptor‐2 (HER2) rabbit mAb (4B5) (Ventana, AZ, USA), anti‐epidermal growth factor receptor (EGFR) mouse mAb (2‐18C19) (pharmDx; Dako), and an anti‐MET (c‐MET) (EP1454Y) rabbit mAb (ab51067; Abcam, MA, USA). Scoring of HER2, EGFR, and MET were carried out based on Hofmann's criteria.23 Positivity was defined when complete or basolateral membrane reveal moderate (2+) or strong (3+) staining in more than 10% of cancer cells.
3
Comprehensive Tumor Histopathology Analysis
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HT-29 tumors were dissected at a size of about 1,000 mm3 and directly transferred to formalin. After fixation the tumors were embedded in paraffin and cut in 4 μm-sections. The sections were stained with hematoxylin and eosin (Biocare Medical) using a Tissue-Tek Prisma Plus, Sakura instrument according to the manufacturer’s instructions. Immunostaining with Anti-Ki67 (DAKO, M7240) and Anti-Annexin V (abcam, ab108321, rabbit) was performed after pretreatment with Targen Retireval Solution, Low pH, Dako, K800521-2; antibody dilution 1: 1000, 30 min RT and cMet (Abcam, ab51067), pretreatment – Target Retrieval Solution, High pH, Dako, K800421-2; antibody dilution 1: 100, overnight incubation then detected with Dako EnVision Flex High pH # K801021-2 kit.
Necrotic areas were quantified by estimating the fraction of necrosis in the whole tumor area in five 10× power fields. Mitotic and apoptotic cells (Annexin V positive) were scored in ten 40× power fields. cMet expression scored according to staining intensity (negative -, weak+, moderate++, or strong+++). The proliferation index was quantified by calculating the fraction of Ki67 positive cells in an 0.25 mm2 grid in a hot spot area. All histopathologic evaluation was done in a blinded manner by a pathologist.
Necrotic areas were quantified by estimating the fraction of necrosis in the whole tumor area in five 10× power fields. Mitotic and apoptotic cells (Annexin V positive) were scored in ten 40× power fields. cMet expression scored according to staining intensity (negative -, weak+, moderate++, or strong+++). The proliferation index was quantified by calculating the fraction of Ki67 positive cells in an 0.25 mm2 grid in a hot spot area. All histopathologic evaluation was done in a blinded manner by a pathologist.
4
Protein Expression and Cell Viability Assay
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Roswell Park Memorial Institute (RPMI)-1640 (Invitrogen, Carlsbad, CA, USA), fetal bovine serum (FBS) (Invitrogen), 1% antibiotic-antimycotic (AA) solution (Invitrogen), Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Invitrogen), 1,4-dithiothreitol (DTT) (Sigma-Aldrich, St. Louise, MO, USA), radioimmunoprecipitation assay (RIPA) buffer (89900; Pierce), bicinchoninic acid assay (23225; ThermoFisher, Waltham, MA, USA), sample buffer (125 mM Tris pH 6.8, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 0.006% bromophenol blue, and 1.8% mercaptoethanol), precast protein gel (4561094; Bio-Rad, Hercules, CA, USA), primary antibodies, anti-c-Met (ab51067; Abcam, Cambridge, UK; 4560; Cell Signaling, Massachusetts, USA), anti-nucleolin (ab22578/ ab13541; Abcam), anti-HER2 (SC-08; Santa Cruz, TX, USA), anti-GAPDH (SC-47724; Santa Cruz), MTS assay solution (G3585; Promega, WI, USA), μ-Slide 8-well (ibid; München, Germany), Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs), and Matrigel (BD Biosciences, NJ, USA) were used.
5
Immunohistochemical Evaluation of Met Expression
6
Western Blot Analysis of Cell Proteins
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Cellular proteins were extracted using radioimmunoprecipitation assay buffer (P0013B, Beyotime Institute of Biotechnology, Haimen, China) was used to extract total protein from the cells. Protein concentration was determined using a bicinchoninic acid assay. The proteins (50 µg/well) were transferred to a polyvinylidene difluoride membrane following electrophoresis with 10% SDS-PAGE. the membrane was incubated for 2 h at room temperature in 5% non-fat dry milk powder. The primary antibodies (rabbit anti-c-Met, ab51067, 1:200 dilution; rabbit anti-E-cadherin, ab76319, 1:200 dilution; both Abcam) were incubated at 4°C overnight, and the secondary antibody Horseradish peroxidase-labeled goat anti-rabbit secondary antibody (ab150077, 1:200, Abcam) was incubated at room temperature for 2 h. An ECL chemiluminescence detection kit (Kangwei Century Biotechnology Co., Ltd., Beijing, China) was used to visualize the protein bands.
7
Immunohistochemical Analysis of Tumor Markers
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The tumor tissue sections (5 µm thick) were made by incision using a microtome (RM2125RT; Leica, Wetzlar, Germany). These were immediately fixed with 4% paraformaldehyde and embedded in paraffin. The standard procedures of Deparaffinization, rehydration, and antigen retrieval were performed. Next, the sections were incubated with the primary antibodies at 4 °C overnight. Then, incubation with secondary antibodies was performed for 30 min at room temperature. After washing with PBS, the sections were stained with diaminobenzidine (DAB, Sigma-Aldrich) and counterstaining was carried out using hematoxylin (Sigma-Aldrich). The pictures were recorded using a light microscope (Olympus BX 51). The primary antibodies Anti-E-cadherin (ab40772, 1/500), anti-N-cadherin (ab18203, 1/1,000), anti-Vimentin (ab92547, 1/500), anti-CD44 (ab157107, 1/1,000), anti-Oct-4 (ab181557, 1/1,000), anti-c-met (ab51067, 1/250), and anti-Nanog (ab109250, 1/250), and secondary antibodies (Goat Anti-rabbit IgG, HRP-linked Antibody, ab205718, 1/2,000) were purchased from Abcam.
8
Western Blot Analysis of Protein Expression
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Total protein was first extracted using SDS-PAGE and then transferred to a PVDF membrane (Thermo, USA). Afterwards, the PVDF membrane was incubated with 1:1000 dilution of antibody: TRIM47 (ab155549, Abcam, USA), BAP1 (ab199396, Abcam, USA), P21 (ab188224, Abcam, USA), P53 (ab131442, Abcam, USA), SRC (ab47405, Abcam, USA), MET (ab51067, Abcam, USA), and c-Myc (ab39688,Abcam, USA). The membrane was washed and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated goat anti-rabbit (Santa Cruz, USA). SuperSignalMT West Puico PLUS (Termo Scientific, USA) was used to develop the blot, using b-actin (ab8226, Abcam, USA) as the loading control. All experiments were performed in triplicate.
9
Western Blot Analysis of NADPH Oxidase Proteins
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Total protein from cells was extracted by lysis and resolved by 8–12% SDS-PAGE. Samples were then electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Boston, MA, USA). PVDF membranes were blocked with 5% BSA or skimmed milk. Target proteins were detected with antibodies against GAPDH (Abcam, ab9485, diluted 1:2500), NOX1 (Abcam, ab78016, diluted 1:1000), NOX2 (Abcam, ab31092, diluted 1:1000), NOX3 (Abcam, ab82708, diluted 1:2000), NOX4 (Abcam, ab133303, diluted 1:2000), NOX5 (Abcam, ab198213, diluted 1:2000), DUOX1 (Abcam, ab178534, diluted 1:5000), DUOX2 (Abcam, ab170308, diluted 1:500), VEGFR-1 (Abcam, ab32152, diluted 1:2000), VEGFR-2 (Abcam, ab11939, diluted 1:1000), VEGFR-3 (Abcam, ab27278, diluted 1:1000), EGFR (Abcam, ab52894, diluted 1:5000), p-EGFR (Abcam, ab40815, diluted 1:2000), PDGFR-α (Abcam, ab203491, diluted 1:1000), PDGFR-β (Abcam, ab32570, diluted 1:10000) and C-Met (Abcam, ab51067, diluted 1:5000) overnight at 2–8 °C, followed by incubation with specific horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, ab6721, diluted 1:10,000) for 2 h at room temperature. Bands were visualized by chemiluminescence with enhanced chemiluminescent detection reagents (Millipore, Boston, MA, USA).
10
Immunoblotting Analysis of Signaling Proteins in Thyroid Cancer
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The protein levels of MET, p-PI3K, PI3K, p-AKT and AKT in thyroid cancer cells were detected by performing immunoblotting assays. Cells were lysed by RIPA buffer (Sigma-Aldrich, USA) with Complete Protease Inhibitor Cocktail (Roche, USA). Cell lysates were transferred to 1.5 mL tube and kept at − 20 °C before use. SDS-PAGE was conducted to separate the cellular proteins. Proteins were loaded onto SDS-PAGE minigel, and then transferred onto PVDF membrane. The blots were probed with the following antibodies at 4 °C overnight: anti-MET (ab51067, Abcam, Cambridge, MA, USA), anti-p-PI3K (phospho Y607, ab182651, Abcam), anti-PI3K (ab86714, Abcam), anti-p-AKT (phospho S473, ab81283, Abcam), anti-AKT (ab179463, Abcam) and anti-GAPDH (ab8245, Abcam) and incubated with HRP-conjugated secondary antibody (1:5000). Signals were visualized using ECL Substrates (Millipore, USA). The protein expression was normalized to endogenous GAPDH.
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