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A novel paper MAP method for rapid high resolution histological analysis.

ABSTRACT: Three-dimensional visualization of cellular and subcellular-structures in histological-tissues is essential for understanding the complexities of biological-phenomena, especially with regards structural and spatial relationships and pathologlical-diagnosis. Recent advancements in tissue-clearing technology, such as Magnified Analysis of Proteome (MAP), have significantly improved our ability to study biological-structures in three-dimensional space; however, their wide applicability to a variety of tissues is limited by long incubation-times and a need for advanced imaging-systems that are not readily available in most-laboratories. Here, we present optimized MAP-based method for paper-thin samples, Paper-MAP, which allow for rapid clearing and subsequent imaging of three-dimensional sections derived from various tissues using conventional confocal-microscopy. Paper-MAP successfully clear tissues within 1-day, compared to the original-MAP, without significant differences in achieved optical-transparency. As a proof-of-concept, we investigated the vasculature and neuronal-networks of a variety of human and rodent tissues processed via Paper-MAP, in both healthy and diseased contexts, including Alzheimer's disease and glioma.

SUBMITTER: Lee M

PROVIDER: S-EPMC8639998 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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A novel paper MAP method for rapid high resolution histological analysis.

Lee Mirae M Woo Jiwon J Kim Doh-Hee DH Yang Yu-Mi YM Lee Eunice Yoojin EY Kim Jung-Hee JH Kang Seok-Gu SG Shim Jin-Kyung JK Park Jeong-Yoon JY

Scientific reports 20211202 1

Three-dimensional visualization of cellular and subcellular-structures in histological-tissues is essential for understanding the complexities of biological-phenomena, especially with regards structural and spatial relationships and pathologlical-diagnosis. Recent advancements in tissue-clearing technology, such as Magnified Analysis of Proteome (MAP), have significantly improved our ability to study biological-structures in three-dimensional space; however, their wide applicability to a variety ...[more]

PMID: 34857810

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Project description:BackgroundComplex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs) from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM) analysis is a new method in which polymerase chain reaction (PCR) and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB) is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB.MethodsHere, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism.ResultsWe were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB.ConclusionsSeveral gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing their association to PTB. It can easily be used for simultaneously analyze any set of SNPs, either as the first choice for new association studies or as a complement to large-scale genotyping analysis. Given that inflammatory pathway is in the base of several diseases, it is potentially useful to analyze a broad range of disorders.

Project description:Morphometry and histology are essential approaches for investigation and diagnosis of musculo-skeletal disorders. Despite the advent of revolutionary methods of image analysis and high resolution three-dimensional imaging technology, basic conventional light microscopy still provides an incisive overview of the structure and tissue dynamics of the musculoskeletal system. This is crucial to both preclinical and clinical research, since several clinically relevant processes, such as bone repair, osteoarthritis, and metabolic bone diseases, display distinct, if not pathognomonic, histological features. Due to the particular characteristics of the skeletal tissues (i.e., the existence of mineralized extracellular matrices), a large number of staining methods applicable to either decalcified or undecalcified tissues are available. However, it is usually the case that several staining methods need to be sequentially applied in order to achieve the different endpoints required to fully assess skeletal tissue structure and dynamics, and to allow morphometric quantification. We describe herein a novel staining method, the RGB trichrome, amenable for application to decalcified, paraffin embedded human musculoskeletal tissues. The acronym RGB corresponds to the three primary dyes used: picrosirius Red, fast Green, and alcian Blue. Although these individual pigments are commonly used either isolated, in binary combinations, or as part of more complex polychrome staining methods, when merged in the RGB trichrome staining produce high-quality/high-contrast images, permitting not only clear identification of different tissues (i.e., the different types of cartilage, bone and fibrous connective tissue), but also discrimination between calcified and uncalcified bone and cartilage, as well as an unexpected diversity of shades of color, while displaying singular properties among polychrome staining methods, such as the unveiling of the bone osteocyte dendritic/canalicular network. Hence, we propose the RGB trichrome as simple but highly-reliable tool for the preclinical and clinical study of the musculoskeletal system.

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A novel paper MAP method for rapid high resolution histological analysis.

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A novel paper MAP method for rapid high resolution histological analysis.

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