Tissue samples from the normal group and the CRC group were fixed and then cut into 4 μm sections for the IHC. In simple terms, the tissue samples were treated with ethylene diamine tetraacetic acid (EDTA) as an antigen extract and then treated with CD31 antibody (1:2000, AB76533, Abcam, Cambridge, MA, USA), CD34 antibody (1:400, AB81289, Abcam, Cambridge, MA, USA), VEGFA antibody (1:400, AB185238, Abcam, Cambridge, MA, USA), m6A antibody (1:200, AB151230, Abcam, Cambridge, MA,USA), METTL3 antibody (1:500, AB195352, Abcam, Cambridge, MA, USA), and IGF2BP1 antibody (1:4000, AB229700, Abcam, Cambridge, MA, USA), separately. These samples were then incubated overnight at 4 °C. Subsequently, the second antibody was incubated at 37 °C for 1 h. Finally, the samples were stained and imaged.
Ab185238
Manufactured by Abcam
Sourced in United States
AB185238 is a mouse monoclonal antibody that recognizes the protein target. The antibody is suitable for use in common laboratory techniques such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab195352, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab151230, by Abcam (1 mentions)
Ab76533, by Abcam (1 mentions)
Ab81289, by Abcam (1 mentions)
Ab229700, by Abcam (1 mentions)
Gapdh, by Zhongshan Golden Bridge Biotechnology (1 mentions)
A2066, by Merck Group (1 mentions)
Anti hif 1α antibody, by Cell Signaling Technology (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Ab6721, by Abcam (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
5 protocols using ab185238
1
Immunohistochemical Analysis of Colorectal Cancer
2
Western Blot Analysis of Cellular Proteins
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Cells were collected and lysed, and the protein concentration was determined. 10% SDS-PAGE gel was added into the protein, and electrophoresis was performed first. After marker separation, 0.45 μm PVDF membrane was transferred between the gel after methanol activated membrane. The protein was separated and transferred to a polyvinylidene difluoride membrane followed by incubation with 5% milk at 20 ± 5 °C for 1 h. The membrane was incubated at 4 °C overnight with the following antibodies: METTL3 (1:1000, AB195352, Abcam, Cambridge, MA, USA), VEGFA (1:1000, AB185238, Abcam, Cambridge, MA, USA), and GAPDH (1:1000, Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China). The secondary antibody was then added. The second antibody was sheep anti-rat and sheep anti-rabbit, and the dilution ratio was 1:5000 and the membrane was incubated at room temperature for 1 h. The protein expression was observed using a chemiluminescence gel imaging system (Tanon 5200, Shanghai, China).
3
Quantitative Protein Expression Analysis by Western Blot
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Western blot was performed as previously described.19 (link) Briefly, total protein was extracted from cultured cells using Laemmili buffer (Sigma-Aldrich; St. Louis, MO, USA) and homogenized for 30 seconds using a VP-50 ultrasonic homogenizer (Taitec corp.; Saitama, Japan) at full power. Soluble proteins were further sonicated for 30 seconds 3 times. The concentrations of protein in the lysates were measured using a Pierce BCA protein assay kit (Thermo Fisher scientific; Waltham, MA, USA). An equal amount of each sample was analyzed by immunoblot analysis, as previously mentioned.20 (link) The following primary antibodies were used: anti-HIF-1α antibody (1:1,000; 36,169; Cell Signaling); anti-VEGF-α antibody (1:1000; ab185238; abcam) and antiactin antibody (1:1,000; A2066; Sigma-Aldrich). The secondary antibodies were horseradish peroxidase-conjugated goat antirabbit and goat antimouse immunoglobulins (Dako, Glostrup, Denmark). Protein signals were detected using ImmunoStar LD (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) or Super Signal West Pico (Thermo Fisher Scientific) chemiluminescence reagents. The intensity of each band was analyzed using ImageJ software (National Institutes of Health; Bethesda, MD, USA).
4
Quantitative Protein Expression Analysis
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Western blot (WB) technique was used in this study to detect the protein expression levels of YTHDF1 and VEGFA. Initially, cell samples were collected and lysed to extract the proteins, and their concentrations were quantified using appropriate methods. Subsequently, the extracted protein samples were loaded onto a 10% SDS-PAGE gel for electrophoretic separation. After completion of electrophoresis, the proteins were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane activated with methanol. The transferred membrane was then incubated in a blocking solution containing 5% milk at a temperature of 20 ± 5 °C for 1 h to prevent nonspecific binding. Following this, the membrane was incubated overnight at 4 °C with specific primary antibodies. The primary antibodies used included anti-YTHDF1 antibody (1:1000, 17479-1-AP, ProteinTech, China), anti-VEGFA antibody (1:2000, AB185238, Abcam, USA), and GAPDH antibody as an internal control (1:1000, 60004-1-Ig, ProteinTech, China). After incubation with the primary antibody, the membrane was washed and incubated with respective secondary antibodies, including goat anti-rabbit and goat anti-mouse antibodies, diluted at a ratio of 1:5000, at room temperature for 1 h. Finally, the protein signals on the membrane were detected using a chemiluminescent gel imaging system (Tanon 5200, Shanghai, China).
5
VEGF Immunohistochemistry in Lung Tissue
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Sections of lung tissue were dewaxed in xylene, dehydrated in graded alcohol, and incubated in 3% H 2 O 2 for 0.5 h. The sections were blocked with normal goat serum, followed by anti-vascular endothelial growth factor (VEGF) (1:100; ab185238, Abcam) antibody incubation. Then the sections were incubated with HRP-labeled goat-anti rabbit IgG (1:1000, ab6721, Abcam) secondary antibody.
Immunohistochemistry was captured by a digital microscope (Nikon Eclipse 80i, Nikon, Tokyo, Japan).
Immunohistochemistry was captured by a digital microscope (Nikon Eclipse 80i, Nikon, Tokyo, Japan).
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