Two-micrometer-thick sections were cut and processed for immunohistochemistry. After being deparaffinized and rehydrated, the sections were immersed in an antigen retrieval bath (Dako, Milan, Italy) for 30 minutes at 98℃ in 1mM of freshly made ethylenediaminetetraacetic acid solution, incubated with 3% H2O2 for 15 minutes in order to quench endogenous peroxidase activity, and then incubated at room temperature for 2 hours with primary antibodies raised against CD34 (Dako) or 1 mg/mL of mouse IgG1 (Dako) as a negative control. This was followed by a 30-minute incubation with the Envision system (Dako). 3,3'-Diamin obenzidinetetrahydrochloride was used as a chromogen to yield brown reaction products. The nuclei were lightly counterstained with hematoxylin solution.
Antigen retrieval bath
Manufactured by Agilent Technologies
Sourced in Italy
The Antigen Retrieval Bath is a laboratory equipment designed to facilitate the process of antigen retrieval. Its core function is to provide a controlled environment for the preparation of tissue samples, allowing for the exposure of target antigens for subsequent immunohistochemical analysis.
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2 protocols using antigen retrieval bath
1
Immunohistochemical Analysis of CD34
2
Immunohistochemical Analysis of SP17 in BC and Testicular Tissue
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2 μm thick-sections were cut from formalin-fixed and paraffin-embedded surgical BC or testicular biopsy tissue and processed for immunohistochemistry. In brief, after deparaffining and rehydration, they were immersed in an antigen retrieval bath (Dako, Milan, Italy), incubated with 3% H2O2 for 15 minutes to quench endogenous peroxidase activity, treated for two hours at room temperature with primary antibodies raised against SP17, or with rabbit IgG (Dako) as a negative control, and then incubated for 30 minutes with the DAKO Envision system (Dako). 3, 3’-diaminobenzidine tetrahydrochloride (Dako) was used as a chromogen to yield brown reaction products. The nuclei were lightly counterstained with hematoxylin solution.
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