Mice tumors were cut into 2-μm sections and embedded with paraffin. Before immunohistochemistry, sections were deparaffinized and rehydrated. Firstly, sections were blocked with 3% H2O2 for 10 min at room temperature followed by incubation with primary antibody against Ki67 and VEGF at 4°C overnight. Secondly, sections were washed with PBS followed by incubation with biotinylated goat anti-mouse immunoglobulin G secondary antibody (Dako, Denmark) at 25°C for 30 min. Thirdly, sections were washed followed by incubation with streptavidin-peroxidase reagent (Dako) and 3, 3ʹ-diaminobenzidine (DAB; Sigma) mixture for 5 min. Finally, sections were counterstained and dehydrated for observation.
Streptavidin peroxidase reagent
Manufactured by Agilent Technologies
The Streptavidin-peroxidase reagent is a protein conjugate used in various immunoassay and detection applications. Streptavidin, a tetrameric protein derived from the bacterium Streptomyces avidinii, is covalently linked to the enzyme horseradish peroxidase. This reagent leverages the high-affinity binding between streptavidin and biotin, a widely used labeling molecule, to facilitate the detection and visualization of target analytes.
Automatically generated - may contain errors
2 protocols using streptavidin peroxidase reagent
1
Immunohistochemical Analysis of Tumor Markers
2
Immunohistochemical Quantification of Proliferation and Apoptosis
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The tumor tissue was fixed in 10% neutral-buffered formalin, embedded in paraffin wax, and cut into sections of 3 μm thickness. Then, the sections were immunohistochemically stained with anti-Ki67 and anti-cleaved (cl)-Caspase-3 antibodies (Cell Signaling Technology) at 4°C overnight. Slides were rinsed with phosphate-buffered saline (PBS) and incubated at room temperature for 30 minutes with biotinylated goat anti-mouse IgG secondary antibody (Dako, Glostrup, Denmark). After washing in Tris-hydrochloride acid buffers (TBS), the slides were incubated with streptavidin-peroxidase reagent (Dako) and treated with 3,3′-diaminobenzidine (DAB; Sigma Aldrich; Merck KGaA) for 5 minutes. Finally, sections were rinsed with ddH2O and counterstained with hematoxylin. Slides were observed under microscope, with the selection of 5 fields of view randomly. The final percentage of positive cells was calculated with the Motic Image software (version 1.2; Micro-Optical Group Co.)
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