Efficient Use of Primary Antibody Labeling for Multiplex Imaging Assays

Use of whole slide imaging systems (WSIs) to perform multiplex imaging assays have enabled the generation of improved, histopathological information from fluorescent labeled tissue biopsies.  The resulting digital images can be analyzed to produce spatial and temporal data which researchers can then link to other “-omics” information sources for developing and refining the understanding of mechanistic pathways.

While the use of multiple fluorescent markers in tissue sections can lead to a wealth of contextual data, the sample preparation techniques often employed by researchers have both time and complexity challenges.  Use of indirect antibody labelling is often used to amplify the fluorescent signal.  This amplification is often a necessity to gain a high enough signal for digital image acquisition.  However, indirect  labeling incurs much longer preparation times as well as additional experiments to ensure the secondary antibody is not prone to non-specific binding or cross-reactivity.  When dealing with three or more fluorescent labels, the sample preparation time can be significant.  For example, three antibodies with direct conjugation can be a half-day protocol versus the multiple days that are often required when using a direct protocol.  Employing direct, primary labeling will shorten this sample preparation time as well as eliminate non-specific binding of any secondary labels; however, the resultant, lower levels of fluorescence staining may not be practical for many WSI systems due to their inability to capture dim signals. 

In newer generation WSI scanners, such as the Axio Scan.Z1 by ZEISS, the incorporation of high dynamic range, 16-bit cameras, improvements in excitation, and focus algorithm advancements make it possible to use direct, primary antibody labeling for multiplex imaging assays.  Not only does this reduce sample preparation times, this enables efficient co-localization of a high number of specific biomarkers to explore complex cell and tissue phenotypes.  The potential of multiplexing techniques are wide-ranging, with a practical application being recent research in liver pathology using fluorescence labeling and WSI to further stratify ductal epithelial diversity (please refer to:  Isse, K. et al.  “Preexisting epithelial diversity in normal human livers: a tissue-tethered cytometric analysis in portal/Periportal epithelial cells.”  Hepatology 2013 Apr;57(4):1632-43.).

For more information on ZEISS Axio Scan.Z1, please visit www.zeiss.com/axioscan