Tissue preparation for immunohistochemical staining may be done by paraffin embedding or freezing. To date, the great majority of IHC studies using phospho-specific antibodies have used formalin-fixed paraffin embedded tissues; however, a limited number of studies have suggested that phospho-specific epitopes may be detected in frozen sections as well. In this study we analyzed a broad panel of phospho-specific and total antibodies directed against various signaling markers on matched fresh frozen or formalin-fixed paraffin embedded xenograft tumors. The results suggest that phospho-epitopes survive fixation and tissue processing, and are not necessarily degraded or dephosphorylated as fixative penetrates. Staining in paraffin-embedded samples matched or was superior to that seen in frozen tissues, though antigen retrieval was often required, including for p-EGFR, p-CREB, p-S6 and p-Histone H3. Determining the optimal fixative for frozen sections was found to be imperative for many antibodies, for example p-erk and p-mTOR. These results will be useful for future studies that are conducted on frozen tissues.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]