TY - JOUR

T1 - A Standardized and Reproducible Workflow for Membrane Glass Slides in Routine Histology and Spatial Proteomics

AU - Nordmann, Thierry M.

AU - Schweizer, Lisa

AU - Metousis, Andreas

AU - Thielert, Marvin

AU - Rodriguez, Edwin

AU - Rahbek-Gjerdrum, Lise Mette

AU - Stadler, Pia-Charlotte

AU - Bzorek, Michael

AU - Mund, Andreas

AU - Rosenberger, Florian A.

AU - Mann, Matthias

N1 - Publisher Copyright: © 2023 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.

PY - 2023

Y1 - 2023

N2 - Defining the molecular phenotype of single cells in situ is key for understanding tissue architecture in health and disease. Advanced imaging platforms have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. Furthermore, high-precision laser microdissection (LMD) of tissue on membrane glass slides is a powerful method for spatial omics technologies and single-cell type spatial proteomics in particular. However, current histology protocols have not been compatible with glass membrane slides and LMD for automated staining platforms and routine histology procedures. This has prevented the combination of advanced staining procedures with LMD. In this study, we describe a novel method for handling glass membrane slides that enables automated eight-color multiplexed immunofluorescence staining and high-quality imaging followed by precise laser-guided extraction of single cells. The key advance is the glycerol-based modification of heat-induced epitope retrieval protocols, termed “G-HIER.” We find that this altered antigen-retrieval solution prevents membrane distortion. Importantly, G-HIER is fully compatible with current antigen retrieval workflows and mass spectrometry–based proteomics and does not affect proteome depth or quality. To demonstrate the versatility of G-HIER for spatial proteomics, we apply the recently introduced deep visual proteomics technology to perform single-cell type analysis of adjacent suprabasal and basal keratinocytes of human skin. G-HIER overcomes previous incompatibility of standard and advanced staining protocols with membrane glass slides and enables robust integration with routine histology procedures, high-throughput multiplexed imaging, and sophisticated downstream spatial omics technologies.

AB - Defining the molecular phenotype of single cells in situ is key for understanding tissue architecture in health and disease. Advanced imaging platforms have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. Furthermore, high-precision laser microdissection (LMD) of tissue on membrane glass slides is a powerful method for spatial omics technologies and single-cell type spatial proteomics in particular. However, current histology protocols have not been compatible with glass membrane slides and LMD for automated staining platforms and routine histology procedures. This has prevented the combination of advanced staining procedures with LMD. In this study, we describe a novel method for handling glass membrane slides that enables automated eight-color multiplexed immunofluorescence staining and high-quality imaging followed by precise laser-guided extraction of single cells. The key advance is the glycerol-based modification of heat-induced epitope retrieval protocols, termed “G-HIER.” We find that this altered antigen-retrieval solution prevents membrane distortion. Importantly, G-HIER is fully compatible with current antigen retrieval workflows and mass spectrometry–based proteomics and does not affect proteome depth or quality. To demonstrate the versatility of G-HIER for spatial proteomics, we apply the recently introduced deep visual proteomics technology to perform single-cell type analysis of adjacent suprabasal and basal keratinocytes of human skin. G-HIER overcomes previous incompatibility of standard and advanced staining protocols with membrane glass slides and enables robust integration with routine histology procedures, high-throughput multiplexed imaging, and sophisticated downstream spatial omics technologies.

U2 - 10.1016/j.mcpro.2023.100643

DO - 10.1016/j.mcpro.2023.100643

M3 - Journal article

C2 - 37683827

AN - SCOPUS:85175029908

VL - 22

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 10

M1 - 100643

ER -