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URL: https://dx.doi.org/DOI:10.17504/protocols.io.36vgre6

Authors: Jessica Riesterer, Erin Stempinski, Claudia Lopez

Group: NCIHTAN

Summary: Tissues, cell monolayers, organoids and xenografts all image differently depending on the sample preparation method utilized. Moreover, the same tissue type from different species may also need different fixation solution and processing methods to have optimal contrast and charge mitigation when the same microscope is used (Borrett & Hughes, 2016; Kizilyaprak, Longo, Daraspe, & Humbel, 2015; Kopek et al., 2017). For example, brain tissue may be processed successfully using a protocol that yields poor images when applied to cancer tissues (unpublished data). Researchers are therefore encouraged to dive into the literature and test new sample preparation protocols for a specific sample-type. Also, and if available, having the ability to evaluate the use of FIB-SEM versus SBF-SEM will help the researcher to design a data collection strategy. The protocols evaluated during development of this workflow included the Dresden protocol (Paridaen, Wilsch-Bräuninger, & Huttner, 2013), Renovo (Mukherjee et al., 2016) and the Hua method (Hua, Laserstein, & Helmstaedter, 2015). We settled on the the Hua method with some modifications as described below, for human cancer biopsies. The final protocol described is sufficient for large format mapping and 3DEM FIB-SEM and SBF-SEM, eliminating the need for multiple samples processed with different protocols. In the case of biopsy tissue, where sample acquisition is limited and precious, flexibility is an important advantage of this workflow.

Affiliations: Oregon Health and Sciences University, Oregon Health and Sciences University, Oregon Health and Sciences University

Version: 1

Publication Date: 2019

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