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View our IHC tissue processing protocol; IHC fixation protocol; sectioning of paraffin-embedded tissue video protocol if you need to prepare your own tissue sections from scratch.
Paraffin-embedded sections are first dewaxed to replace the wax with water; this is because most staining solutions are aqueous. For more detailed information please see our IHC deparaffinization protocol.
Dewaxing is usually followed by an antigen retrieval method, which unmasks antigens that have been obscured during the fixation process. It is crucial to find the most suitable antigen-retrieval method for the target you are interested in. You can choose from the two methods of antigen retrieval: heat-mediated (also known as heat-induced epitope retrieval or HIER) and enzymatic in IHC antigen retrieval protocol.
Next, depending upon the type of antibodies that will be used, a series of blocking steps take place that serve to minimize non-specific interactions of the antibody and block endogenous enzymes that can result in false positives.
After blocking, the sections are incubated in antibodies; this incubation can be in directly conjugated primaries or can consist of sequential incubations in both primary and secondary antibodies (fluorescent or enzyme-linked).
If an enzyme-linked secondary is used, a further incubation in substrate will be required, otherwise the sections can be mounted and imaged.
View our IHC staining protocol for paraffin, frozen and free floating sections; IHC staining protocol for whole mount samples for more detailed information.