Optimal Formalin-Fixed Paraffin-Embedded Sample Preservation for Efficient Staining in Multiplex Imaging

The accuracy of immunohistochemical techniques depends critically on the preservation of antigenic integrity in tissue samples. Although formalin-fixed paraffin-embedded tissues are commonly used for long-term storage, several studies reported signal degradation over time. However, the extent of this degradation and the influence of storage conditions on a wide range of markers remain poorly characterized. In this study, tissue blocks collected over an 8-year period and tissue sections stored under different conditions (room temperature [RT], 4 °C, -20 °C, and -80 °C) were analyzed. Twenty-five antibodies targeting immune, stromal, and structural markers were used to stain 4 different tissue sections. Samples were acquired using imaging mass cytometry. Signal intensities were first quantified, then the data were analyzed, before and after normalization, using the imcRtools pipeline. A gradual loss of signal intensity was observed in blocks stored at RT for more than 6 years, affecting the detection of certain markers regardless of tissue type. For some sections, significant signal degradation was observed after only 1 week at RT and for others after 1 month of storage. Markers such as CD20, CD45, and CD45RA proved to be particularly sensitive. Storage at -20 °C or -80 °C preserved the quality of the staining, whereas storage at 4 °C only allowed partial preservation. Normalization corrected some variations but did not enable the recovery of signals that were severely altered or absent. Commonly used RT storage causes a gradual reduction in imaging mass cytometry--detected immunoreactivity, which may affect scientific interpretation and diagnosis. Storing sections at -20 °C is, therefore, an effective and accessible solution. For retrospective studies, we recommend using blocks that are less than 6 years old. Overall, these results highlight the importance of preestablished standardized preservation protocols to ensure the reproducibility of analyses.