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.
Peroxidase conjugated complex
Manufactured by Agilent Technologies
The Peroxidase-conjugated complex is a laboratory reagent used in various analytical techniques. It consists of an enzyme, peroxidase, covalently attached to a carrier molecule. This complex is designed to facilitate the detection and quantification of target analytes in samples through enzymatic reactions.
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2 protocols using peroxidase conjugated complex
1
Immunohistochemical Analysis of Cell Proliferation and Apoptosis
2
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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