![]() ![]() Include a control slide without antibody and fluorophore to check for autofluorescence in your tissue (see: IHC-Controls). Autofluorescence of the tissue may hamper interpretation of results especially in green- and red-channel fluorophores. In Immunofluorescence, use fluorophores with narrow emission spectra, narrow bandpass filters in your microscope to avoid bleed-through, and target the less abundant protein with the brighter fluorophore. Spectral Differentiation of Colors and Compatibility of Chromogenic Substrates When including a heat denaturation step in your protocol, consider taking a heat-resistant substrate, e.g. In chromogenic IHC-P, heat or low pH-buffers may be used to strip antibodies from the first IHC-P round when only primary antibodies from the same species and isotype are available. Monoclonal antibodies from the same host species but with different isotypes can be detected using isotype-specific secondary antibodies. When using indirect IHC methods for antibody-antigen detection (see: Secondary-systems), the primary antibodies should ideally originate from different host species to avoid cross-reaction of the secondary systems. However, Multiplexing has many technical challenges, so several important issues should be considered: Host Species of Primary Antibodies Multiplex IHC enables the detection of two or more targets on one slide and is particulaly valuable when single IHC-P is not sufficient to accurately show the anticipitated result: e.g. immunophenotyping of cells using two or more markers. Multiplex IHC-P can be done using either fluorophores or chromogenic substrates. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |