Xenograft tumors were fixed in 10% formalin and paraffin-embedded. Next, 2-μm sections were deparaffinized and subjected to antigen retrieval according to standard procedures. The sections were incubated with Ki-67 (clone MIB-1; Dako; dilution 1: 100) for 30 min. Then, the sections were incubated with secondary antibodies and visualized with 3, 3′-diaminobenzidine. Ki-67 immunoreactivity was evaluated using a labeling index (% of positive cells).
Ki 67 clone mib 1
Manufactured by Agilent Technologies
Sourced in United States, Denmark, Germany, United Kingdom, Japan
Ki-67 (clone MIB-1) is a monoclonal antibody used in immunohistochemistry to detect the Ki-67 protein, a marker of cellular proliferation. It is commonly used in research to assess the growth fraction of a cell population.
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Lab products found in correlation
Cd20 clone l26, by Agilent Technologies (6 mentions)
Her2 clone 4b5, by Roche (5 mentions)
Benchmark ultra, by Roche (4 mentions)
Bond max autostainer, by Leica (4 mentions)
Cd10 clone 56c6, by Leica (4 mentions)
Bcl2 clone 124, by Agilent Technologies (4 mentions)
Bond max, by Leica (4 mentions)
Benchmark xt, by Roche (4 mentions)
Myc clone y69, by Abcam (3 mentions)
P53 clone do 7, by Agilent Technologies (3 mentions)
Mum1 clone mum1p, by Agilent Technologies (3 mentions)
Cd20 clone l26, by Leica (3 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Benchmark xt system, by Roche (2 mentions)
Cd31 clone jc70a, by Agilent Technologies (2 mentions)
Chromogranin a, by Agilent Technologies (2 mentions)
Cd56 clone 1b6, by Leica (2 mentions)
Herceptest, by Agilent Technologies (2 mentions)
Pr clone pgr 636, by Agilent Technologies (2 mentions)
Er clone sp1, by Lab Vision (2 mentions)
97 protocols using ki 67 clone mib 1
1
Quantifying Xenograft Tumor Proliferation
2
Quantifying Tumor Cell Proliferation and Apoptosis
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The xenograft sections were processed and stained with an antibody against Ki-67 (clone MIB-1, 1:100; Dako) for the analysis of mitotically active cells. Apoptosis was assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL; Abcam) according to the manufacturer’s instructions. The images were digitally recorded at a magnification of 400× with an Olympus BX53 microscope and an Olympus DP27 camera. Areas from the digitalized color photomicrographs were analyzed. Two examiners independently determined the percentage of positive cells.
3
Quantitative Immunohistochemical Analysis of Autophagy Markers
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The slides were incubated with the primary antibodies (1:100 dilution, rabbit monoclonal antibody (mAb) for ATG7, ab 52472, Abcam, UK; 1:500 dilution, rabbit mAb for LC3, 3868 CST, USA; 1:200 dilution, rabbit mAb for Beclin-1, 3395 CST, USA; and 1:75 dilution, ki-67, clone MIB-1, Dako, Santa Clara, CA, US) overnight at 4 °C and with Anti-rabbit Envision+/horseradish peroxidase (HRP) (Dako) or Anti-mouse Envision+/HRP (Dako) secondary antibody for 1 h. Staining of ATG-7, LC3, and Beclin-1 was categorised semi-quantitatively based on the percentage of positive tumour cells: 0 (5% positive cells), 1 (6–25% positive cells), 2 (26–50% positive cells), 3 (51–75% positive cells), and 4 (>75% positive cells). Cytoplasmic and membrane staining intensities were also determined semi-quantitatively as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). Sections’ scores were defined as the extent of staining × intensity, while the Ki-67 score was calculated as the percentage of positively stained cells among the total number of malignant cells scored. The scoring was conducted using three high-power (×400) fields in the invasive edge of the tumour, which represents the spectrum of staining observed in the whole section.
4
Immunohistochemical Assessment of ER and Ki-67
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Immunohistochemical analysis was performed on 4-mm sections obtained from formalin-fixed paraffin-embedded material. The primary antibodies used for the evaluation were antiestrogen receptor alpha (clone SP1) from Ventana Medical Systems (Tucson, USA) provided as a ready-to-use antibody and Ki-67 clone Mib-1 obtained from Dako (Glostrup, Denmark) applied at a concentration of 1 : 100. The sections were dewaxed and subjected to an antigen retrieval protocol within a BenchMark Ultra instrument (Ventana Medical Systems) followed by incubation with primary antibodies. Bound antibodies were visualized using the streptavidin–biotin–peroxidase method and diaminobenzidine as the chromogen (ultraView Kit, Ventana Medical System). For positive controls, tissue microarray sections from breast carcinoma cases were used. As a negative control we incubated tissue sections with commercially obtained IgG-antibodies of the same subclass as the monoclonal antibodies used for Ki-67/MiB1 and ERalpha.
5
Immunohistochemical Profiling of Neuroendocrine Tumors
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Immunohistochemistry was performed on formalin fixed tissue sections using the following antibodies at Mayo Clinic: OSCAR cytokeratin (clone OSCAR, predilute, BioLegend, Dedham, MA), chromogranin A (clone LK2H10, predilute, Ventana, AZ), CDX2 (clone EPR2764Y, 1/200, Cell Marque, Rocklin, CA), Islet 1 (clone 1H9, 1/800, abcam, Cambridge, MA), INSM1 (clone A8, 1/100, Santa Cruz, CA), Ki-67 (clone MIB-1, 1/20, Dako, Carpinteria, CA) and TTF-1 (clone SPT24, 1/100, Leica, Newcastle, UK). INSM1 and chromogranin were used as neuroendocrine markers. First, the sections were deparaffinized then rehydrated and stained online using antibody specific epitope retrieval techniques with the Ventana Benchmark XT system (Ventana, AZ).
Immunohistochemistry was performed at the University of Virginia Health System for T-PIT using the TBX19 antibody (clone T-PIT, 1/2000, Atlas Antibodies AB, Sweden) on the Ventana Benchmark platform. Immunohistochemistry for all markers was scored as follows: Negative (−) = 0% of cells staining and Positive (+) = > 10% of cells staining as an arbitrary minimum value. Automated Ki-67 analysis was performed using the digital method previously published by Kroneman et al. [18 (link)].
Immunohistochemistry was performed at the University of Virginia Health System for T-PIT using the TBX19 antibody (clone T-PIT, 1/2000, Atlas Antibodies AB, Sweden) on the Ventana Benchmark platform. Immunohistochemistry for all markers was scored as follows: Negative (−) = 0% of cells staining and Positive (+) = > 10% of cells staining as an arbitrary minimum value. Automated Ki-67 analysis was performed using the digital method previously published by Kroneman et al. [18 (link)].
6
Immunohistochemical Profiling of PD-L1, Immune Markers
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Immunohistochemistry (IHC) was performed on archival, formalin-fixed, paraffin-embedded tissues, using the following antibodies: PD-L1 (clone 123 C3SP142, Ventana-Diapath, dilution 1:50), CD3 (clone 2GV6, Ventana-Diapath, dilution RTU), Cytokeratin 20 (clone SP33, Ventana, dilution RTU), Chromogranin (clone LK2H10, Ventana, dilution RTU), TTF-1 (clone 847G3/1, Ventana, dilution RTU), and Ki67 (clone MIB-1, Dako, dilution 1:100). PD-L1 positivity was assessed on ICs and tumor cells (TCs), as previously described by Lipson et al. [34 (link)].
7
Immunohistochemical Biomarker Analysis
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IHC staining for ER (clone 6F11, Novocastra, Leica Biosystems), PgR (clone 1A6, Novocastra), Ki67 (clone MIB-1, Dako, Glostrup, Denmark) and c-erbB2 (HER2/neu, A0485 polyclonal antibody, Dako) on each slide was performed as previously described,14 (link) while detection of cyclin D1 (clone SP4, Spring Bioscience, USA), phosphatase and tensin homolog (PTEN) (clone 6H2.1, code M3627, Dako) and mammalian target of rapamycin (mTOR) (clone 49F9, code 2976, Cell Signaling Technology, Danvers, Massachusetts, USA) proteins was performed as mentioned in earlier published studies.2 15 (link)
8
Lymph Node Immunohistochemistry Profiling
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Lymph nodes were formalin‐fixed and paraffin‐embedded (FFPE). Paraffin sections were immunostained for CD21 (clone 1F8), CD23 (clone MHM6), Bcl‐2 (clone 124), CD10 (clone 56C6), Ki‐67 (clone Mib‐1) (Dako, Denmark), Stathmin (clone SP49; Spring Bioscience, Pleasanton, CA USA), VCAM (Clone VCAM1/843; Scytek Laboratories, Logan, UT, USA), and CXCL13 (Polyclonal Goat; R&D Systems, Minneapolis, MA, USA), using an automated immunostainer (Dako). The staining of follicular stroma and interfollicular fibroblastic stroma was graded quantitatively as previously described 20 : 0, absent; 1+ focal; 2+ extensive; 3+ diffuse.
9
Immunohistochemistry and EBER Analysis of LBCL
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Immunohistochemistry and EBER in situ hybridization were done upon diagnosis using an automated platform (Benchmark ULTRA, Ventana Medical Systems, Tucson, USA). The following antibodies were routinely applied: CD20 (clone L26, prediluted, Ventana Medical Systems, Tucson, USA), CD79 (clone SP19, prediluted, Ventana Medical Systems, Tucson, USA), CD5 (clone SP19, prediluted, Ventana Medical Systems, Tucson, USA), Ki67 (clone Mib-1, dilution 1:100, DAKO, Leiden, the Netherlands), CD10 (clone SP67 prediluted, Ventana Medical Systems, Tucson, USA), BCL2 (clone 124, prediluted, Ventana Medical Systems, Tucson, USA), BCL6 (clone GI191E/A8, prediluted, Ventana Medical Systems, Tucson, USA), MUM1 (clone MRQ 43, prediluted, Ventana Medical Systems, Tucson, USA), C-MYC (clone Y69, prediluted, Ventana Medical Systems, Tucson, USA), and INFORM EBER (Epstein-Barr virus early RNA) Probe (Cat. Nr. 800-2842, Ventana Medical Systems, Tucson, USA).
All LBCLs were classified according to the WHO classification of tumors of the hematopoietic and lymphoid tissues (2008) and in addition were subdivided into germinal center (GCB) and non-germinal center (non-GCB) subtypes [2 , 16 (link)]. A cutoff of 40% C-MYC and 50% BCL2 positive lymphoma cells was used to identify double expressors [9 (link), 12 (link)].
All LBCLs were classified according to the WHO classification of tumors of the hematopoietic and lymphoid tissues (2008) and in addition were subdivided into germinal center (GCB) and non-germinal center (non-GCB) subtypes [2 , 16 (link)]. A cutoff of 40% C-MYC and 50% BCL2 positive lymphoma cells was used to identify double expressors [9 (link), 12 (link)].
10
Proliferation Fraction in Glioblastoma
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Deparaffinized GBM tissue from primary surgery or biopsy was used to evaluate the proliferation fraction of tumour cells (tissue slices of thickness 4 μm). Antigen retrieval was performed using 10 mM Tris/1 mM EDTA (pH 9) in a microwave at 700 W. Endogenous peroxidase and biotin were blocked using routine techniques. The slides were incubated with the primary antibody, Ki67 (clone MIB-1; Dako, Glostrup, Denmark) at room temperature for 1 h, followed by application of the secondary antibody, peroxidase-conjugated rabbit anti-mouse serum (Dako), and the tertiary antibody, peroxidase-conjugated goat anti-rabbit serum (Dako), for 30 min each. The first antibody was diluted 1/100 in 1% bovine serum albumin (BSA)/phosphate-buffered saline (PBS). The secondary and tertiary antibodies were diluted 1/100 in 1% BSA/PBS with 1% AB serum. Colour was developed with 3,3′-diaminobenzidine (Sigma, Zwijndrecht, The Netherlands) for 10 min. The slides were scanned for hot spots of proliferative activity. In one high-power field (×400 magnification) the fraction of Ki67-positive nuclei/total number of nuclei was determined.
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