Problem of Antigen Retrieval Standardization

Duration of fixation, especially for formalin-sensitive epitopes 

Type of microwave oven and heating parameters, other heating sources, or other epitope retrieval treatments such as enzyme digestion and ultrasound 

Tested or new antibodies obtained from the same clone or not 

Source of antibodies even similar monoclonal antibodies obtained from different sources may show differential affinity for the epitope 

Variable staining methods automated immunostainers or manual staining 

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