Will your antibodies work in IHC-F?

IHC-F can refer to several different protocols in different sample types. Tissue that is formalin fixed and paraffin embedded is used with dye-conjugated antibodies and this can be IHC-F. IHC-F may also refer to fluorescent detection of primary antibody OR dye-conjugated antibody in formaldehyde fixed frozen tissue. Both of these sample types are routinely validated by their respective applications. Finally IHC-F in the context of this article refers to chromogenic detection of an antibody in frozen tissue and this is not something that we routinely do.

We have found that antibodies recommended for IHC on paraffin embedded samples may work on fresh frozen samples as well. Because antibody performance in a given application is influenced by how the samples are prepared, antibodies that are known to work in immunocytochemistry (IF-IC) may be more likely to work in fresh frozen tissues, as the samples are handled more similarly.

When we do test our antibodies for use in frozen tissue with chromogenic detection, we begin by optimizing the fixative for a given antibody, as we have seen over a number of comparisons that the choice of fixative can impact staining results. We generally compare the following fixatives: acetone, 10% neutral buffered formalin, 3-4% formaldehyde, and 3-4% formaldehyde followed by a methanol incubation. Once the fixative has been optimized we perform a small titration of the primary antibody to determine the optimal dilution.

Last updated: September 16, 2024