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The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for chromogenic IHC experiments using paraffin-embedded tissue samples.
This protocol provides a basic guide for the fixation, microtome sectioning, and staining of paraffin-embedded tissue samples. Each investigator must determine the precise experimental conditions required to generate a strong and specific signal for each antigen of interest. If R&D Systems primary antibodies are employed, please refer to the product data sheets to obtain the recommended working dilutions. In this protocol, signal visualization is achieved using R&D Systems Cell and Tissue Staining Kits. For all other reagents, please follow the manufacturer’s instructions.
Please read protocol in its entirety before beginning.
This staining protocol has been developed and optimized for use with R&D Systems Cell and Tissue Staining Kits.
Note: Excessive fixation may result in the masking of an epitope and strong non-specific background signal that can obscure specific labeling. If necessary, an antigen retrieval protocol can be performed at this time.
Note: Endogenous peroxidase and biotin can react with secondary reagents and cause non-specific background staining. R&D Systems Cell and Tissue Staining Kits contain reagents to address these potential artifacts (applied in protocol steps 3 – 7).
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