Tumor specimens were prepared as described above. Subsequently, the tissue sections were deparaffinized and placed in antigen retrieval solution (Abcam) for 15 min at 100°C. Following incubation in 1% bovine serum albumin (Sigma-Aldrich) for 30 min, primary antibodies [YAP, CD31, cyclinA, cyclinD1, cyclinE; dilution, 1:200] were applied to the slides for 2 h at 37°C. Following washing with PBS, the sections were additionally incubated with the HRP-conjugated donkey anti-goat secondary antibody (dilution, 1:2,000; ab205723; Abcam) and avidin-conjugated horseradish peroxidase (43–4423; Thermo Fisher Scientific, Inc.). Finally, the sections were treated with 3,3′-diaminobenzidine, and counterstained with hematoxylin, and dehydrated. A negative control was performed by replacement of primary antibody with PBS. Two pathologists reviewed the results, with IHC staining intensity determined by Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
Ab205723
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab205723 is a monoclonal antibody that recognizes a specific epitope. It is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab53519, by Abcam (4 mentions)
Ab205719, by Abcam (4 mentions)
Ab8245, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Bandscan 5.0 system, by Bio-Rad (2 mentions)
Ecl chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Sds page, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Antigen retrieval solution, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab207802, by Abcam (1 mentions)
Ab175449, by Abcam (1 mentions)
Ab179695, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab9722, by Abcam (1 mentions)
Ab263899, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab40772, by Abcam (1 mentions)
8 protocols using ab205723
1
Immunohistochemical Analysis of Tumor Biomarkers
2
Quantification of NLRP3 Inflammasome Proteins
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The expression of NLRP3, ASC, caspase‐1, IL‐1β, and TGF‐β1 was measured by Western blot analysis. Total proteins in ventricular chamber tissues were isolated using radioimmunoprecipitation assay buffer, and concentrations were measured using BCA Protein Assay Kits (Beyotime Institute Biotechnology). Total protein was separated by 10% polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Nonspecific binding was blocked with 5% skim milk in Tris‐buffered saline Tween‐20, then the membranes were incubated for 4 h at 37°C with primary antibodies against NLRP3 (ab263899, 1:1000), ASC (ab175449, 1 μg/ml), caspase‐1 (ab207802, 1:1000), IL‐1β (ab9722, 0.1 μg/ml), TGF‐β1 (ab179695, 1:1000), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (ab181602, 1:10000) (Abcam), followed by the corresponding secondary antibodies (ab205718/ab205723, Abcam) for 1 h. The membranes were rinsed, then proteins were visualized using enhanced chemiluminescence. Relative protein expression was calculated as the ratio of target protein expression to that of the reference protein (GAPDH). The experiment was repeated three times and the data were averaged.
3
Quantitative Protein Analysis of Tumor Cells
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Total proteins in tumor tissues or different cell lines were extracted using RIPA lysis buffer (P0013B, Beyotime), and the protein concentration was evaluated using a BCA assay kit (23,250, Pierce, MA, USA). Then 25 μg of total protein was separated in SDS‐PAGE gels (P0052A, Beyotime), and further transferred onto the PVDF membranes (FFP32, Beyotime). Subsequently, the membranes were soaked in 5% skimmed milk for 2 h and further incubated with primary antibodies against KNG1 (1:3000, ab97761, 48 kD, Abcam, CA, USA), SNAIL1 (1:2000, ab53519, 29kD, Abcam), TWIST1 (1:1000, ab50887, 21kD, Abcam), Vimentin (1:5000, ab92547, 54kD, Abcam), ZEB1 (1:2000, ab180905, 124kD, Abcam), N‐Cadherin (1:2000, ab18203, 130kD, Abcam), E‐Cadherin (1:10000, ab40772, 97kD, Abcam), ZO1 (1:3000, ab96587, 187kD, Abcam), CDC25A (1:1000, ab989, 59kD, Abcam), cyclin D1 (1:1000, ab134175, 34kD, Abcam), CDKN2A (1:4000, ab201980, 17kD, Abcam), and GAPDH (1:1000, 36kD, ab8245, Abcam). The next day, the membranes were then incubated with Goat Anti‐Mouse (1:5000, ab205719, Abcam), Goat Anti‐Rabbit (1:5000, A0208, Beyotime), and Donkey Anti‐Goat (1:5000, ab205723, Abcam) secondary antibodies for 1 h. Finally, the protein signal in the membranes was detected under Image Lab system 3.0 (Bio‐Rad; Hercules) after the membranes were incubated with detection buffer (P0272, Beyotime) for 1 min.
4
Western Blot Analysis of EMT Markers
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Total proteins were extracted from the cells using RIPA buffer (Beyotime, China) and protein concentration was determined using a Bio-rad DC Assay kit (Hercules, USA). Equal amounts of protein (20 μg) were separated on 10% SDS-PAGE (Invitrogen) and electroblotted onto a PVDF membrane (Merck, Germany), which was then blocked by 5% non-fat milk for 1 h and further incubated with E-cadherin (E-Cad, Cat#14472, 1:1000, CST, USA), N-Cadherin (N-Cad, Cat #14215, 1:1000, CST, USA), Snail (ab53519, 1:1000, Abcam, Cambridge, MA, USA), and GAPDH (ab8245, 1:2000, Cambridge, MA, Abcam) overnight at 4°C. Secondary antibodies (donkey anti-goat IgG H&L (HRP) (ab205723, 1:2000, Cambridge, MA, Abcam) or goat anti-mouse IgG H&L (HRP) (ab205719, 1:2000, Cambridge, MA, Abcam)) were incubated with the membrane for 2 h. The membrane was developed by ECL chemiluminescence (Thermo Scientific, USA) and Band Scan 5.0 system (Bio-Rad). GAPDH served as an internal reference.
5
Immunohistochemical Detection of FZD2 in Tissue
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IHC staining was performed to detect FZD2 expression in tissue samples. The standard streptavidin–biotin–peroxidase complex method was used for IHC staining. Briefly, after blocking of endogenous peroxidase activity in tissue sections with 3% H2O2 and antigen retrieval with a target retrieval solution (S1699; Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturer's instructions, the sections were incubated with 10% normal goat serum (S-1000; Vector Laboratories, Burlingame, CA, USA) in PBS for 30 min. Next, sections were incubated with a primary anti-FZD2 antibody (ab109094, 1:200, Abcam, Cambridge, UK) at 4°C overnight. After three washes with PBS, the sections were incubated with donkey anti-goat IgG H&L (HRP) (ab205723, 1:2000, Abcam, Cambridge, UK) for 30 min at room temperature. Finally, sections were incubated with a peroxidase substrate solution (Sk-4100, Vector Laboratories, Burlingame, CA, USA) until the desired staining intensity was attained. Sections were rinsed with tap water, counterstained with haematoxylin, and mounted with coverslips. The results of IHC staining were viewed and scored separately by two experienced pathologists. The expression levels of FZD2 expression were assessed and scored as follows: negative (0; complete absence of staining), weak staining (score: 1), moderate staining (score: 2), or strong staining (score: 3).
6
Histological Analysis of Ghrelin in GI Tissue
7
Western Blot Analysis of EMT Markers
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Cells were lysed on ice using RIPA buffer (Beyotime, Shanghai, China). The proteins were harvest and their concentration was determined by BCA Assay Kit (Pierce Chemical Co., Rockford, IL, USA). The proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, Billerica, MA, USA), which were sealed by 5% skimmed milk for 1 h. Then, the membranes were incubated with the following primary antibodies at 4°C overnight: E-Cadherin (CST, #14,472, 1:1000, USA), N-Cadherin (CST, #14,215, 1:1000, USA), and Snail (Abcam, ab53519, 1:2000, UK). After washing, the membranes were incubated with secondary goat anti-rabbit (Abcam, ab205719, 1:1000, UK) and donkey anti-goat (Abcam, ab205723, 1:2000, UK) for 2 h at room temperature. After rinsing, the membranes were visualized by ECL reagent (Cell Signaling Technology, USA) and examined by Amersham Imager 600 (GE Healthcare Life Sciences). The data were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
8
Western Blot Analysis of EMT Markers
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Total proteins were extracted from cells using RIPA buffer (Beyotime, China), the protein concentration was determined by BCA protein assay kit (Pierce, Germany). The protein (20 μg) were separated by 10% SDS-PAGE (Invitrogen, USA) and transferred onto PVDF membranes (Merck, Germany). The membranes were blocked by 5% nonfat milk for one hour and then incubated with the following primary antibodies overnight at 4°C: E-cadherin (E-Cad, Cat#14472, 1:1000, CST, USA), N-Cadherin (N-Cad, Cat #14215, 1:1000, CST, USA), Snail (ab53519, 1:1000, Abcam, USA), GAPDH (ab8245, 1:2000, Abcam, USA). After that, the membranes were further incubated with secondary antibodies, donkey anti-goat IgG H&L (HRP) (ab205723, 1:2000, Abcam, USA), goat anti-mouse IgG H&L (HRP) (ab205719, 1:2000, Abcam, USA) for two hours, and visualized by ECL chemiluminescence (Thermo Scientific, USA) using a Band Scan 5.0 system (Bio-Rad, Hercules, USA). GAPDH served as a control.
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