Serial 4 μm thick paraffin sections were used for immunohistochemistry with antibodies specific for IGF2BP3 (ab179807, Abcam, USA, 1:100), E2F3 (DF12390, Affinity Biosciences, Changzhou, China, 1:50), and Ki67 (AF1738, Beyotime, Shanghai, China, 1:50). A streptavidin peroxidase (SP) immunohistochemical kit (Maixin, Fuzhou, China) was used according to the instructions. Images were taken with a Nikon microscope (Eclipse Ci, Nikon Ltd, Japan). Image-Pro Plus 6.0 was employed to analyse the levels of protein expression by calculating the values of mean integrated optical density (integrated optical density/area) for statistical analysis [47 (link)].
Ab179807
Manufactured by Abcam
Sourced in United States
Ab179807 is a recombinant monoclonal antibody that recognizes the protein ZRANB3. It is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab139509, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Af1738, by Beyotime (1 mentions)
Sp immunohistochemical kit, by Maixin Group (1 mentions)
Eclipse ci, by Nikon (1 mentions)
Ab7817, by Abcam (1 mentions)
Ab195352, by Abcam (1 mentions)
Ab309096, by Abcam (1 mentions)
Ab8827, by Abcam (1 mentions)
Rip kit, by Merck Group (1 mentions)
Ab185656, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Af7021, by Affinity Biosciences (1 mentions)
Bca protein concentration determination kit, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Vazyme (1 mentions)
Ba1054, by Boster Bio (1 mentions)
β actin, by Proteintech (1 mentions)
Ab195377, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab252346, by Abcam (1 mentions)
8 protocols using ab179807
1
Immunohistochemical Analysis of Protein Expression
2
Western Blotting Antibody Characterization
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Western blotting was conducted by using the following antibodies: rabbit anti-IGF2BP3 (ab179807, 1:500, Abcam), rabbit anti-PLAGL2 (ab139509, 3:1,000, Abcam), rabbit anti-METTL3 (ab195352, 1:500, Abcam), rabbit anti-METTL14 (ab309096, 1:400, Abcam), and rabbit anti-β-actin (ab8827, 1:1,000, Abcam). The β-actin (ab7817, 1:2,000, Abcam) was employed as the internal control. This image was captured using a gel imaging system (Odyssey, LI-COR Biosciences, USA).
3
RNA Immunoprecipitation and qRT-PCR Analysis
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4
Localization of IGF2BP3 in EC Cells
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EC cells on glass coverslips were incubated with specific antibodies against IGF2BP3 (ab179807, Abcam, 1:80) overnight at 4 °C and then incubated with Alexa Fluor 488 goat anti-rabbit IgG (Affinity Biosciences, Changzhou, China, 1:200). To detect colocalization, we treated the cells with FISH probes and the anti-IGF2BP3 primary antibody in hybridization buffer at the same time. Images were captured by a confocal laser scanning microscope (Examiner. Z1, Carl Zeiss, Germany).
5
Western Blot Analysis of Protein Targets
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The cells were harvested and lysed on ice with RIPA lysis buffer (Beyotime, Shanghai, China). The BCA protein concentration determination kit (Beyotime, Shanghai, China) was used to determine the protein concentration. After boiling for 10 min, proteins were separated by 10% SDS–PAGE and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membrane was subsequently blocked for 1 h in 5% fat-free milk, incubated with the primary antibodies for 1.5 h at 25 °C, and then incubated with the horseradish peroxidase-labeled secondary antibody (Zhongshan Jinqiao, Beijing, China, 1:3000) for 1 h at 25 °C. After repeated washing with TBST, the membranes were developed by an enhanced chemiluminescence kit (Vazyme, Nanjing, China). The primary antibodies used were as follows: GAPDH (AF7021, Affinity Biosciences, Changzhou, China, 1:3000), IGF2BP3 (ab179807, Abcam, USA, 1:1000), and E2F3 (27615-1-AP, Proteintech, Wuhan, China, 1:500).
6
Immunohistochemistry Protocol for IGF2BP3 and PLAGL2
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IHC was performed following the previously described protocol [12 (link)]. Paraformaldehyde was used to fix the tissue samples, followed by embedding them in paraffin. Antigen retrieval for IHC analysis was achieved through heat-induced treatment with ethylenediaminetetraacetic acid (EDTA) buffer. To inhibit endogenous peroxidase activity, 3% H2O2 was utilized. The sections were then blocked with 5% goat serum for a duration of 1 h, followed by incubation with antibodies targeting IGF2BP3 (ab179807, 1:200, Abcam, MA, USA) or PLAGL2 (ab139509, 1:200, Abcam). Following this, the sections were subjected to a 1-h incubation period with the secondary antibody (BA1054, 1:1,500, Boster, China). Afterwards, the sections underwent processing using an ABC HRP kit and a DAB substrate kit (Zhongshan Jinqiao Biotechnology Co., Ltd, China). Nuclei were counterstained with hematoxylin. Subsequently, the sections were washed with water to eliminate any excess substrate, dehydrated, and finally covered with a mounting medium. The evaluation of images was performed using the IHC profiler plugin in ImageJ (NIH, USA).
7
Whole Cell Lysates Preparation and Immunoblotting
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Whole cell lysates were collected in RIPA lysis buffer (Beyotime, China). The antibodies used in this study are IGF2BP3 (#ab179807, Abcam), and β-actin (#20536-1-AP, Proteintech) antibodies. HRP-conjugated anti-rabbit secondary antibody was used for the ECL detection.
8
Western Blot Analysis of m6A Regulators
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Proteins were harvested and dissolved in RIPA lysis buffer, and protein concentrations were detected by enhanced Bicinchoninic Acid (BCA) protein assay kit (Beyotime, China). And the equivalent amounts of protein were separated by SDS-PAGE on 10% acrylamide gels at 60 v for 2.5 h and transferred to PVDF membranes under a constant current of 340 mA for 1.5 h. Quantitative analysis was performed by ImageJ. The primary antibodies were anti-ALKBH5 (ab195377, Abcam, MA, USA), anti-METTL3 (86132S, CST, MA, USA), anti-YTHDF1 (ab252346, Abcam, MA, USA), anti-IGF2BP3 (ab179807, Abcam, MA, USA), anti-FTO (31687S, CST, MA, USA), and anti-GAPDH (ab8227, Abcam, MA, USA). The secondary antibodies were goat polyclonal anti-Rabbit-IgG (14708S, CST, MA, USA).
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