Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

in Science translational medicine by Pablo A Valdes, Chih-Chieh Jay Yu, Jenna Aronson, Debarati Ghosh, Yongxin Zhao, Bobae An, Joshua D Bernstock, Deepak Bhere, Michelle M Felicella, Mariano S Viapiano, Khalid Shah, E Antonio Chiocca, Edward S Boyden

TLDR

  • dExPath is a new way to look at cells in the brain that have been affected by disease. It uses a special microscope to separate proteins from each other, making it easier to see the cells and their structures. The study found that dExPath can reveal previously unseen structures and disease markers, which could help doctors diagnose and treat brain diseases more accurately.

Abstract

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

Overview

  • The study presents an expansion microscopy (ExM) variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. The primary objective of the study is to improve antibody access to epitopes in these specimens, which may render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

Comparative Analysis & Findings

  • The study compares the outcomes observed under different experimental conditions or interventions detailed in the study. The results show that dExPath-expanded specimens reveal previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. The study also shows that dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence.

Implications and Future Directions

  • The study's findings suggest that dExPath may improve the accuracy and sensitivity of immunohistochemistry, which could have significant implications for clinical science and diagnosis. Future research could explore the use of dExPath in other types of tissues and pathologies, as well as the development of new antibodies that can take advantage of the improved epitope access provided by dExPath.
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