Total proteins were extracted using RIPA Lysis Buffer (Beyotime) and next separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and incubated with the blocking buffer. Subsequently, the membranes containing protein bands were probed with the primary antibodies against Ki67 (ab231172; Abcam, Cambridge, MA, USA), Proliferating cell nuclear antigen (PCNA; ab18197; Abcam), CSF-1 (ab99178; Abcam) and GAPDH (ab9485; Abcam) at 4 °C overnight and subsequently treated with the secondary antibodies (ab205718; Abcam). The protein bands were imaged using an enhanced chemiluminescence kit (ECL; Beyotime). Each sample was in triplicate, and three independent experiments were conducted.
Ab231172
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab231172 is a recombinant antibody product. It is designed to detect the target protein in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (3 mentions)
Ab18197, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab111691, by Abcam (1 mentions)
Ab109461, by Abcam (1 mentions)
Ab181603, by Abcam (1 mentions)
Ab34277, by Abcam (1 mentions)
Ab263947, by Abcam (1 mentions)
Ab207086, by Abcam (1 mentions)
Ab133543, by Abcam (1 mentions)
Ab200738, by Abcam (1 mentions)
Ab134157, by Abcam (1 mentions)
Ab76315, by Abcam (1 mentions)
6 protocols using ab231172
1
Protein Quantification and Western Blot
2
Immunohistochemical Analysis of Tumor Markers
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The harvested xenograft model nude mice tumors were fixed and embedded in paraffin. The tumor tissues were sliced, deparaffinized, and hydrated, then the endogenous peroxidase and non-specific proteins were blocked, and the primary antibody was added and incubated at 4 °C Overnight. The primary antibodies include anti-ki-67 (Abcam, ab231172, Cambridge, UK), anti-KMT5A (Abcam, ab111691, Cambridge, UK). Next, the slices are washed with PBS and incubated with secondary antibody (Abcam, ab205718, Cambridge, UK) at 37 °C for 30 min, developed with DAB, counterstained with hematoxylin, dehydrated, and observed under an optical microscope.
3
Immunohistochemical Analysis of Ki-67 in Xenograft Tumors
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Xenograft tumors derived from nude mice were immobilized using formalin then enveloped with paraffin. Later, a paraffin section (5‐μm thick) was prepared24 and immunostained with anti‐Ki‐67 (ab231172; Abcam) at 4°C overnight, then probed with secondary antibody (ab205718; Abcam) at indoor temperature for 1 h. Subsequently, sections were dyed with diaminobenzidine and hematoxylin in succession, followed by observation using a light microscope (magnification: 200×) (Olympus).
4
Assessing Tumor Growth in Immunodeficient Mice
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Specific pathogen-free female immunodeficient mice (3~5 weeks old, 18~21 g) were obtained from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. [SCXK(Zhejiang)2019-0004]. U87 cells (4×106) were transfected with sh-CMTM6 or sh-NC and injected into each nude mouse. Tumor growth was measured every 7 days. Tumor volume was calculated as (length × width2)/2. Finally, the mice were euthanized and tumors were removed and weighed. The tumor tissues were incubated with anti-Ki-67 antibodies (1:500, ab231172, abcam). 3 fields were selected to calculate the ratio of positive tumors and staining intensities. Animal experiments were performed under a project license [No. SYXK(SHU)2017-0044] granted by ethics committee board of The Third Affiliated Hospital of Soochow University, in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines for the care and use of animals.
5
Protein Expression Analysis in Cellular Aging
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After removing the culture medium, 500 μl protein lysate is added to each well for lysis for 30 minutes, centrifuged at 4° C, 1500 r/min for 5 minutes. And the supernatant is taken to detect the protein concentration AGING of the sample. Transfer the protein to PVDF membrane through Gels preparation, samples loading, electrophoresis and wet transfer and close the blocking solution for 1 hour. Sequentially, primary antibody SHP2 (Abcam, ab187040, 1:5000), Ki-67 (Abcam, ab231172, 1:5000), p-STAT1 (Abcam, ab109461, 1:1000), p-STAT3 (Abcam, ab76315, 1:1000), p-STAT5 (Abcam, ab278764, 1:1000), p-STAT6 (Abcam, ab263947, 1:1000), IL-4 (Abcam, ab34277, 1:1000), IL-10 (Abcam, ab52909, 1:1000), Cathepsin-L (Abcam, ab200738, 1:1000), Cathepsin-S (Abcam, ab134157, 1:1000), Cathepsin-K (Abcam, ab207086, 1:1000), Arginase-1 (Abcam, ab133543, 1:1000) and GAPDH (Abcam, ab181603, 1:10000) are added and the secondary antibody is incubated for 2 hours at room temperature. Exposure imaging is performed and Quantity One V4 software is used for analysis.
6
Xenograft Tumor Growth Inhibition
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Animal experiment was authorized by the Institutional Animal Care and Use Committee of Shanxi Provincial Cancer Hospital and performed based on the guidelines of the China Science Review of Laboratory Animal Welfare (GB/T 35892-2018). The BALB/c nude mice were obtained from Vital River Laboratory (Beijing, China) and then assigned to four groups (6 mice per group). The A549/DDP cells (2 × 107) transfected with sh-circHUWE1 or control were implanted into the right back near the forelimb of BALB/c nude mice. Furthermore, BALB/c nude mice in the DDP groups were treated with 5 mg/kg of DDP every week by intraperitoneal injection after 7 d injection, with phosphate-buffered saline as control. The volume of xenograft tumor was calculated according to the equation: volume = 1/2 (length × width2). The xenograft was resected for weight measurement and then stored for further research. For immunochemistry for Ki-67 and TNFAIP8, a study was conducted as previous description [26 (link)] using Ki-67 (ab231172; 1 : 100 dilution; Abcam) and TNFAIP8 immunochemistry kit (#Yaji Biological, Shanghai, China).
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