Frozen tissue preparation for high-resolution multiplex histological analyses of human brain specimens
The availability of a comprehensive tissue library is essential for elucidating the function and pathology of human brains. Considering the irreplaceable status of the formalin-fixation-paraffin-embedding (FFPE) preparation in routine pathology and the advantage of ultra-low temperature to preserve nucleic acids and proteins for multi-omics studies, these methods have become major modalities for the construction of brain tissue libraries. Nevertheless, the use of FFPE and snap-frozen samples is limited in high-resolution histological analyses because the preparation destroys tissue integrity and/or many important cellular markers. To overcome these limitations, we detailed a protocol to prepare and analyze frozen human brain samples that is particularly suitable for high-resolution multiplex immunohistological studies. As an alternative, we offered an optimized procedure to rescue snap-frozen tissues for the same purpose. Importantly, we provided a guideline to construct libraries of frozen tissue with minimal effort, cost and space. Taking advantage of this new tissue preparation modality to nicely preserve the cellular information that was otherwise damaged using conventional methods and to effectively remove tissue autofluorescence, we described the high-resolution landscape of the cellular composition in both lower-grade gliomas and glioblastoma multiforme samples. Our work showcases the great value of fixed frozen tissue in understanding the cellular mechanisms of CNS functions and abnormalities.
KeywordsFrozen glioma tissue Oligodendrocyte precursor cell (OPC) Multiplex immunofluorescence staining Brain tissue library Autofluorescence.
We appreciate the technique support from the Human Brain Bank of Zhejiang University. This work is supported by the National Key Research and Development Program of China, Stem Cell and Translational Research (2016YFA0101201 to C.L.), the Science Foundation for Distinguished Young Scientists of Zhejiang Province (LR17H160001 to C.L.), the National Science Foundation of China (81673035 to C.L.), the Thousand Talent Program for Young Outstanding Scientists, China (to C.L.) and Science funding of Zhejiang Province (LY17H160016 to H.S.).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
Informed consent was obtained from all individual participants included in the study.
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