The endpoint xenograft tumors were isolated, fixed in formalin, and paraffin embedded. Tissue sections were processed for immunostaining and incubated overnight with p-AMPKα (T172) (40H9) (#2535, 1:200 dilution), Ki-67 (D2H10) (#9027, 1:200 dilution), or cleaved caspase-3 (Asp175) (5A1E) (#9664, 1:400 dilution) antibodies (Cell Signaling Technology). Next, the sections were counterstained with hematoxylin and subjected to IHC analysis as described previously (8 (link)).
Ki 67 d2h10
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
Ki-67 (D2H10) is a primary antibody product offered by Cell Signaling Technology. It is designed for the detection of the Ki-67 protein, which is a cellular marker for proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3 asp175 5a1e, by Cell Signaling Technology (2 mentions)
Foxm1, by Cell Signaling Technology (1 mentions)
Asf1b, by Cell Signaling Technology (1 mentions)
Plenti cmv puro luciferase, by Addgene (1 mentions)
Tfam d5c8, by Cell Signaling Technology (1 mentions)
Pgc 1α, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab16669, by Abcam (1 mentions)
Ab228462, by Abcam (1 mentions)
Ultravision detection system, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated igg antibody, by Cell Signaling Technology (1 mentions)
Atp7a, by Abcam (1 mentions)
5 protocols using ki 67 d2h10
1
Immunohistochemical Analysis of Xenograft Tumors
2
Quantitative Protein Expression Analysis
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TMAs were kindly provided by the Neuropathology lab of HSM-CHULN. Protein levels were assessed by immunohistochemical (IHC) staining with UBE2C antibody (Boston Biochem, Cat# A650), ASF1B (Cell Signaling Technology, Cat# 2902), FoxM1 (Cell Signaling Technology, Cat# 5436)or Ki-67 (D2H10) (Cell Signaling Technology, Cat# 9027). For UBE2C, ASF1B and FoxM1 we used semi-quantitative scores of intensity (low or high staining intensity) and frequency (low: 0%-49% staining or high: 50%-100% staining), performed by two independent researchers and validated by a pathologist. For Ki67, an automated software was used to quantify the percentage of positive nuclei (ImmunoRatio).
3
Stable Isogenic Cell Lines from Mutant p53 Variants
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Unless otherwise mentioned, all cell lines were obtained from American Type Culture Collection and used at early passage. Mutant p53 (R175H and R273H) overexpression plasmids encoding proline at codon 72 of p53 were a generous gift from Bert Vogelstein from Johns Hopkins School of Medicine. These plasmids were subjected to site-directed mutagenesis to generate R72 variants (Quick Change II, Stratagene), and all plasmids were verified by sequencing. Cells were transfected with the constructs of interest (5 µg of DNA) with Fugene and selected in 800 µg/mL neomycin for 14 d to make stable isogenic cell lines. For the luciferase plasmids, H1299 and PC3 cells were labeled with luciferase by lentiviral infection by using the plasmids pLenti-CMV-Puro-Luciferase (Addgene) and pLenti-UbC-RedFLuc-T2A-Puro (Targeting Systems, LP-30). The pLenti-UBc-RedFLuc-T2A-Puro plasmid was a generous gift from Dr. Meenhard Herlyn of The Wistar Institute. The antibodies used were as follows: p53 (DO-1) (Calbiochem, OP-43), GAPDH (Cell Signaling Technology, 2118S), TFAM (D5C8) (Cell Signaling Technology, 8076), PGC-1α (EMD Millipore, ST1202), PPARγ (R&D Systems, PP-A3409A-00), and Ki67 (D2H10) (Cell Signaling Technology, 9027).
4
Immunohistochemistry for Immune Profiling
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Tissue samples were collected and stained followed the manufacturer’s protocol. In brief, deparaffinized and rehydrated sections were treated for antigen retrieval using sodium citrate buffer, blocked with normal goat serum for 30 min at room temperature, and then incubated with primary antibody against CD3 (ab16669, Abcam, Cambridge, UK), PD-L1(ab228462, Abcam), Ki67(D2H10, Cell Signaling Technology, Danvers, MA) at 4 °C overnight. Slides were incubated with secondary antibodies, counterstained with hematoxylin, and visualized by the Ultra Vision Detection System (Thermo Fisher Scientific). Signal intensity was scored by two independent observers who were blind to the experimental groups.
5
Antibody Validation for Protein Analysis
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SLC31A1/CTR1 antibody (anti-rabbit, polyclonal; cat. no. GTX48534) was purchased from GeneTex. ATP7A (anti-rabbit, polyclonal; ID product code ab125137) was purchased from Abcam. ATP7B (anti-rabbit, polyclonal; cat. no. NB100-360) was purchased from Novus Biologicals. Cleaved caspase-3 (Asp175) (5A1E) (anti-rabbit, monoclonal; product no. 9664), Ki-67 (D2H10) (anti-rabbit, monoclonal; product no. 9027), and horseradish peroxidase (HRP)-conjugated IgG antibody (goat anti-rabbit, monoclonal; product no. 7074) were purchased from Cell Signaling Technology, Inc.
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