Immunohistochemistry was performed, and diagnoses were confirmed. Percentages of positive cells were assessed and assigned to one of the following parameters: negative ≤10%, and ≥10% positive. Representative tissue blocks were cut at 4 mm thickness, deparaffinized with xylene rinse, and rehydrated with distilled water through graded alcohol. IHC staining was conducted on the other four slides using a two-step procedure. Slides were then incubated overnight at 4°C in humidified chambers with human Ki-67 (Abcam, Cambridge, UK; diluted 1:200), P-Akt (Cell Signaling Technology; diluted 1:200), and cleaved caspase-3 (Asp175; diluted 1:100; Cell Signaling Technology). Slides were washed thrice with PBS and further incubated with secondary antibodies for 30 min at room temperature. After washing with PBS, immunolabeled sections were incubated with biotin-conjugated secondary antibody for 20 min at room temperature, then with peroxidase-conjugated complex (Dako) for 20 min, visualized with 3,3′-diaminobenzidine, counterstained with hematoxylin, and then examined by light microscopy.
Human ki 67
Manufactured by Abcam
Sourced in United Kingdom, Japan
Human Ki-67 is a protein that is commonly used as a marker for cellular proliferation. It is expressed during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in resting cells (G0). The Ki-67 protein is a widely accepted and reliable tool for assessing the growth fraction of a given cell population.
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4 protocols using human ki 67
1
Immunohistochemical Analysis of Cell Proliferation and Apoptosis
2
Immunohistochemical Analysis of Tumor Samples
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Blood smear slides were fixed with methyl alcohol and dried for Giemsa staining. Lung, liver, or tumor was excised from NOG mice inoculated with or without cell lines. Part of the excised organs were fixed with dry-ice acetone, embedded in Tissue-Tek OCT compound (Sakura Finetechnical Co. Ltd., Tokyo, Japan), and stored at −80 °C until use. Frozen sections of the organs were prepared using a Cryostat and were fixed in acetone at room temperature for 20 min. The rest of the excised organs were fixed with 4 % paraformaldehyde for H&E staining. These organs were also stained with antibodies against human OPN (O-17, IBL, Gunma, Japan), human CD4 (MT310, DAKO), human IL-2Rα (CD25) (R&D Systems, Inc., Minneapolis), mouse OPN (O-17, IBL, Gunma, Japan), human Ki-67 (Abcam), and mouse fibroblast activation protein (FAP) (rabbit polyclonal, Abcam). Tissue samples from patients with ATL were analyzed at Tohoku University Hospital. This study was approved by the Ethics Committees of Tohoku University Hospital. The tissues were also stained with H&E, human OPN (MPIIIB101, DSHB), human FAP (rabbit polyclonal, Abcam), human CD25 (4C9, Roche), and a macrophage marker CD68 (PG-M1, DAKO) as described above.
3
Immunohistochemical Analysis of Ki-67
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Tissue samples were fixed in 10% formalin (10%) and prepared as 4 μm thick paraffin sections. These paraffin sections were subjected to heat-induced epitope repair, incubated with human Ki-67 (1:200, Abcam), and observed under a light microscope by two pathologists to assess proteins’ cellular localization and immunostaining levels.
4
Immunohistochemical Analysis of Tissue Samples
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3 mm thick, from representative tissue blocks were cut, deparaffinised with xylene rinse and rehydrated with distilled water through graded alcohol. Immunohistochemical staining was performed on the other four slides using the two-step procedure. The slides were then incubated overnight at 4°C in humidified chambers with human Ki-67 (Abcam; diluted 1:200), cleaved-caspase-3 (Cell signaling; diluted 1:200), cleaved-PARP rabbit monoclonal antibody (Cell signaling; diluted 1:100) and anti-human LC3 rabbit monoclonal antibody (Cell signaling; diluted 1:500) were used. The slides were washed three times in a phosphate buffered solution and further incubated with a secondary antibody for 30 min at room temperature. After washing in phosphate buffered solution, the immunolabeled sections were incubated with biotin-conjugated secondary antibody for 20 min at room temperature, then with peroxidase-conjugated complex (Dako) for 20 min, and finally visualized with 3, 3′-diaminobenzidin and counterstained with hematoxylin, and then examined by light microscopy.
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