Separate a small part of the tissue specimen and shape it in a customized mold for chip production and fix it overnight in 4% paraformaldehyde (PFA). Tissue blocks were embedded in paraffin in a prepared array. Then the sample was sliced (5 μm) and adhered to a poly-L-lysine coated glass slide for immunohistochemical staining, which was performed as previously described52 (link),53 (link), using specific antibody against N-MYC (1:600 dilutions; Cell Signaling Technology 51705) and Aurora kinase A (1:200 dilutions; Abcam ab52973). Blindly, with no knowledge of the clinicopathological characteristics of the tumour, the immunoreactivity in tissue sections was observed under three microscopes at random and then evaluated by 3 pathologists. Differences in scoring were discussed until a consensus was reached. The tissue sections were then scored under an optical microscope according to the degree of staining (0 ∼ 3 points were negative staining, light yellow, light brown, dark brown) and the positive range (1 ∼ 4 points were 0 ∼ 25%, 26 ∼ 50%, 51 ∼ 75%, 76 ∼ 100%). Finally, samples were divided into a high-expression group and a low-expression group based on the median of the final staining score. All procedures adhered to the ethical standards set by the Clinical Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (Approval No: XHEC-D-2016-037 ).
Ab52973
Manufactured by Abcam
Ab52973 is a primary antibody targeting an unspecified antigen. It is intended for use in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Sc 56, by Santa Cruz Biotechnology (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab469, by Abcam (1 mentions)
Ab75987, by Abcam (1 mentions)
Ab32085, by Abcam (1 mentions)
Ab235958, by Abcam (1 mentions)
Ab122769, by Abcam (1 mentions)
Ab103232, by Abcam (1 mentions)
Ab183506, by Abcam (1 mentions)
Ab150075, by Abcam (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Ab181861, by Abcam (1 mentions)
Ab202291, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Nonfat milk, by Solarbio (1 mentions)
Western blotting detection kit ecl human igg, by Solarbio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
7 protocols using ab52973
1
Immunohistochemical Evaluation of Tumor Markers
2
Cell Lysis and Western Blot Analysis
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The standard protocol (described above) for cell lysis and western blot was followed with exception that the membrane washes were in tris-buffered saline with 0.1% Tween 20 (TBS-T) and the membrane blocking and antibody probing was performed with 5% BSA in TBS-T. The primary antibodies used for protein detection were rabbit monoclonal anti-Aurora A (1:10,000, ab52973, Abcam), mouse monoclonal anti-PCNA (1:4,000, PC10, sc-56, Santa Cruz Biotechnology), rabbit monoclonal anti-phospho-histone H3 (pSer10) (1:10,000, 04-817, Merck) and rabbit polyclonal anti-histone H3 (1:10,000, ab1791, Abcam). The secondary antibodies were goat anti-rabbit and goat anti-mouse (31460 and 31430, Thermo Scientific) diluted 1:10,000 in TBS-T containing 5% BSA. The immunoblot quantification was performed by ImageJ software (NIH) and the data for normalization to the loading control were obtained following the integration of the band intensity. Unprocessed immunoblot scans for AURKA expression are provided in Supplementary Fig. 9 .
3
Immunohistochemical Detection of AURKA
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Tumor tissue was embedded in paraffin and sectioned for subsequent immunohistochemistry for AURKA detection. After deparaffinization, hydration, antigen extraction, and blockage of sections, primary antibodies were added and incubated at 4°C overnight. Samples were then incubated with secondary antibody following manufacturer's instructions. Subsequently, DAB solution was added dropwise, followed by 10 min of incubation. Hematoxylin was utilized for 1 min of counterstain and then stopped staining. Photographs were taken under a microscope (Nikon, Japan). Anti‐AURKA was purchased from Abcam (ab52973).
4
Aurora A Expression in Colorectal Cancer
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Colorectal cancer tissue samples were collected at Zhongshan Hospital. The study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University (B2018-276) in accordance with the Helsinki Declaration. Immunohistochemical staining was performed on TMAs containing 233 CRC tissue samples. For each sample, two cores from different locations were included in the array. A 4-μm section of TMAs was used for immunohistochemistry and stained with monoclonal anti-Aurora A antibody (Abcam#ab52973) as described previously (Carvalho et al., 2009 (link)). Aurora A staining was evaluated on the basis of the frequency of positive nuclei, and intensity was divided into four groups: negative (<10%), weak (≥10% and <25%), moderate (≥25% and <50%) or strong (≥50%) (Belt et al., 2012 (link); Dotan et al., 2012 (link)). All samples were grouped by two independent observers in a blinded manner. If disagreement between the two observers occurred, a third observer was consulted, and the majority group was noted.
5
Progesterone Receptor Positive Endometrial Cancer
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We collected a total of 182 progesterone receptor positive human endometrial tissue samples, 107 stage III-IV cancer tissue of which had accompanying follow-up information, and 75 cancer-adjacent endometrial tissue samples from archives of paraffin-embedded tissues between May, 2011 and May, 2014 at the Department of Pathology of Peking Union Medical College Hospital. The follow-up was performed until May 30, 2019. The pathological diagnoses were reconfirmed by a pathologist. The project was approved by the Ethical Committee (Peking Union Medical College Hospital), and informed consent was acquired from patients or family members. IHC was performed as previously described (22 (link)). Anti-antibody (AKT1 1:250, Abcam, ab235958; PR 1:100, Abcam, ab32085; PBK 1:100, Abcam, ab75987; BIRC5 1:800, Abcam, ab469; AURKA 1:100, Abcam, ab52973; GTSE1 1:50, Abcam, ab103232; KNSTRN 1:50, Abcam, ab122769; PSMB10 1:100, Abcam, ab183506) was used for IHC. The scoring details have been described previously (23 (link)).
6
Immunofluorescence Analysis of AURKA
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Cells were seeded in 8-well chamber slides (3 × 104 cells/well) treated with MLN8237 or transfected with AURKA siRNA and then were washed with PBS and fixed with 50% methyl alcohol and 50% acetone at 4 °C for 30 min. Next, the cells were blocked with 1% BSA for 1 h at 37 °C. Thereafter, the cells were incubated with the primary AURKA antibody (abcam; ab52973; 1:500) diluted with blocking buffer at 4 °C overnight. Next, the cells were washed with PBS three times and incubated with the second antibody (abcam; ab150075; 1:200) for 1 h at 37 °C. The blue-fluorescent DAPI nucleic acid stain (Invitrogen; D1306) was used as a counterstain. The slides were mounted with coverslips, and the cells were visualized with a fluorescence microscope.
7
Western Blot Analysis of Protein Signaling
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Cells were lysed with RIPA lysis buffer (Beyotime), and protein concentrations were determined by BCA. The samples were separated by SDS-PAGE. Gel proteins were transferred to PVDF membranes (Millipore), sequentially blocked with 5% nonfat milk (Solarbio), and then incubated with primary antibodies overnight at 4 °C. PVDF membranes were further incubated with secondary antibodies, and chemiluminescent detection was performed using the Western Blotting Detection Kit ECL (Human IgG) (Solarbio). Primary antibodies used for western blot included p-Aurora-A (host species: Rat, #3079, CST), Aurora-A (host species: Rat, #ab52973, Abcam), GAPDH (host species: Rabbit, #ab181602, Abcam), PFKFB3 (host species: Rat, # ab181861, Abcam), p-PFKFB3 (host species: Rat, # ab202291, Abcam), p-ERK (host species: Rat, #4370, CST), ERK (host species: Rat, #4695, CST), p-AKT (host species: Rat, #a4060, CST), and AKT (host species: Rat, #4685, CST). GST (host species: Rat, # ab252882, Abcam). ImageJ software was performed to quantify the band intensities of Western blot figures.
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