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The Journal of Molecular Diagnostics :... Jan 2012In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of...
In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays.
Topics: Biomarkers; Cell Line, Tumor; Fixatives; Formaldehyde; Gene Expression; Humans; In Situ Hybridization, Fluorescence; Lymphocytes; Palatine Tonsil; Paraffin Embedding; RNA; Sensitivity and Specificity
PubMed: 22166544
DOI: 10.1016/j.jmoldx.2011.08.002 -
The EMBO Journal Jan 2018Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity...
Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.
Topics: Animals; COS Cells; Chlorocebus aethiops; Drosophila melanogaster; Fixatives; Formaldehyde; Glyoxal; HeLa Cells; Humans; Immunohistochemistry; Mice; Microscopy, Fluorescence; Nerve Tissue Proteins; Tissue Fixation
PubMed: 29146773
DOI: 10.15252/embj.201695709 -
Methods (San Diego, Calif.) Nov 2012We describe a method, Hi-C, to comprehensively detect chromatin interactions in the mammalian nucleus. This method is based on Chromosome Conformation Capture, in which...
We describe a method, Hi-C, to comprehensively detect chromatin interactions in the mammalian nucleus. This method is based on Chromosome Conformation Capture, in which chromatin is crosslinked with formaldehyde, then digested, and re-ligated in such a way that only DNA fragments that are covalently linked together form ligation products. The ligation products contain the information of not only where they originated from in the genomic sequence but also where they reside, physically, in the 3D organization of the genome. In Hi-C, a biotin-labeled nucleotide is incorporated at the ligation junction, enabling selective purification of chimeric DNA ligation junctions followed by deep sequencing. The compatibility of Hi-C with next generation sequencing platforms makes it possible to detect chromatin interactions on an unprecedented scale. This advance gives Hi-C the power to both explore the biophysical properties of chromatin as well as the implications of chromatin structure for the biological functions of the nucleus. A massively parallel survey of chromatin interaction provides the previously missing dimension of spatial context to other genomic studies. This spatial context will provide a new perspective to studies of chromatin and its role in genome regulation in normal conditions and in disease.
Topics: Animals; Cells, Cultured; Chromatin; Chromosome Mapping; Cross-Linking Reagents; DNA; DNA Fragmentation; Epistasis, Genetic; Fixatives; Formaldehyde; Gene Library; Genome, Human; Humans; Nucleic Acid Conformation; Sequence Analysis, DNA; Tissue Fixation
PubMed: 22652625
DOI: 10.1016/j.ymeth.2012.05.001 -
Journal of Visualized Experiments : JoVE Jul 2012The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue,...
The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate (5,7). Often, changes in response to hypoxia begin before the tissue can be preserved (12). The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures (3). It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.
Topics: Animals; Fixatives; Formaldehyde; Perfusion; Polymers; Rats; Tissue Fixation
PubMed: 22871843
DOI: 10.3791/3564 -
PloS One 2013The pericardial tissue is commonly used to produce bio-prosthetic cardiac valves and patches in cardiac surgery. The procedures adopted to prepare this tissue consist in...
BACKGROUND
The pericardial tissue is commonly used to produce bio-prosthetic cardiac valves and patches in cardiac surgery. The procedures adopted to prepare this tissue consist in treatment with aldehydes, which do not prevent post-graft tissue calcification due to incomplete xeno-antigens removal. The adoption of fixative-free decellularization protocols has been therefore suggested to overcome this limitation. Although promising, the decellularized pericardium has not yet used in clinics, due to the absence of proofs indicating that the decellularization and cryopreservation procedures can effectively preserve the mechanical properties and the immunologic compatibility of the tissue.
PRINCIPAL FINDINGS
The aim of the present work was to validate a procedure to prepare decellularized/cryopreserved human pericardium which may be implemented into cardiovascular homograft tissue Banks. The method employed to decellularize the tissue completely removed the cells without affecting ECM structure; furthermore, uniaxial tensile loading tests revealed an equivalent resistance of the decellularized tissue to strain, before and after the cryopreservation, in comparison with the fresh tissue. Finally, immunological compatibility, showed a minimized host immune cells invasion and low levels of systemic inflammation, as assessed by tissue transplantation into immune-competent mice.
CONCLUSIONS
Our results indicate, for the first time, that fixative-free decellularized pericardium from cadaveric tissue donors can be banked according to Tissue Repository-approved procedures without compromising its mechanical properties and immunological tolerance. This tissue can be therefore treated as a safe homograft for cardiac surgery.
Topics: Animals; Compliance; Cryopreservation; Fixatives; Humans; Immunocompetence; Implants, Experimental; Materials Testing; Mice; Pericardium; Stress, Mechanical; Tissue Engineering
PubMed: 23705010
DOI: 10.1371/journal.pone.0064769 -
Progress in Retinal and Eye Research Sep 2015Immunohistochemical and ex vivo anatomical studies have provided many glimpses of the variety, distribution, and signaling components of vertebrate retinal neurons. The... (Review)
Review
Immunohistochemical and ex vivo anatomical studies have provided many glimpses of the variety, distribution, and signaling components of vertebrate retinal neurons. The beauty of numerous images published to date, and the qualitative and quantitative information they provide, indicate that these approaches are fundamentally useful. However, obtaining these images entailed tissue handling and exposure to chemical solutions that differ from normal extracellular fluid in composition, temperature, and osmolarity. Because the differences are large enough to alter intercellular and intracellular signaling in neurons, and because retinae are susceptible to crush, shear, and fray, it is natural to wonder if immunohistochemical and anatomical methods disturb or damage the cells they are designed to examine. Tissue fixation is typically incorporated to guard against this damage and is therefore critically important to the quality and significance of the harvested data. Here, we describe mechanisms of fixation; advantages and disadvantages of using formaldehyde and glutaraldehyde as fixatives during immunohistochemistry; and modifications of widely used protocols that have recently been found to improve cell shape preservation and immunostaining patterns, especially in proximal retinal neurons.
Topics: Fixatives; Formaldehyde; Humans; Immunohistochemistry; Osmolar Concentration; Retina; Retinal Neurons; Staining and Labeling; Tissue Fixation
PubMed: 25892361
DOI: 10.1016/j.preteyeres.2015.04.001 -
Clinical Neuropathology 2016An optimal fixative should ideally combine the advantages of formalin fixation and freezing, allowing for good preservation of histology and molecular components, easy...
An optimal fixative should ideally combine the advantages of formalin fixation and freezing, allowing for good preservation of histology and molecular components, easy handling and storage, lack of toxicity, and low costs. Most of these criteria are fulfilled by ethanol-based solutions, and due to our good experience with the commercial RCL2 fixative, reflected by our published single-center trial, we initiated a multicenter ring trial. However, during its course, RCL2 was discontinued on the market. Therefore, we created our own agent, KINFix, composed of the same main constituents as RCL2, and employed it in our laboratory with similar results. Here we present our evaluation of the three fixatives formalin, RCL2, and KINFix from the perspective of histopathology as well as nucleic acid and protein analyses in comparison to fresh frozen tissues together with the multicenter ring trial data for RCL2. We observe that RCL2 and KINFix offer comparable histomorphology and superior template for molecular analyses than formalin. Moreover, KINFix as freely available fixative might overcome some of the difficulties related to the commercial agents. Therefore, we conclude that KINFix might be an attractive complement to formalin in tissue processing and advocate its use in neuropathological practice.
Topics: Animals; Fixatives; Formaldehyde; Humans; Immunohistochemistry; Nucleic Acids; Paraffin Embedding; Tissue Fixation
PubMed: 26521938
DOI: 10.5414/NP300907 -
Archives of Pathology & Laboratory... Feb 2012
Topics: Fixatives; Formaldehyde; Humans; Lung Neoplasms; Neoplasm Proteins; Tissue Fixation
PubMed: 22288957
DOI: 10.5858/arpa.2011-0363-LE -
The Journal of Histochemistry and... Apr 2017
Topics: Buffers; Ethanol; Fixatives; Formaldehyde; Tissue Fixation
PubMed: 28347267
DOI: 10.1369/0022155416687278 -
Biomolecules May 2021Mitochondria are highly dynamic organelles, constantly undergoing shape changes, which are controlled by mitochondrial movement, fusion, and fission. Mitochondria play a...
Mitochondria are highly dynamic organelles, constantly undergoing shape changes, which are controlled by mitochondrial movement, fusion, and fission. Mitochondria play a pivotal role in various cellular processes under physiological and pathological conditions, including metabolism, superoxide generation, calcium homeostasis, and apoptosis. Abnormal mitochondrial morphology and mitochondrial protein expression are always closely related to the health status of cells. Analysis of mitochondrial morphology and mitochondrial protein expression in situ is widely used to reflect the abnormality of cell function in the chemical fixed sample. Paraformaldehyde (PFA), the most commonly used fixative in cellular immunostaining, still has disadvantages, including loss of antigenicity and disruption of morphology during fixation. We tested the effect of ethanol (ETHO), PFA, and glutaraldehyde (GA) fixation on cellular mitochondria. The results showed that 3% PFA and 1.5% GA (PFA-GA) combination reserved mitochondrial morphology better than them alone in situ in cells. Mitochondrial network and protein antigenicity were well maintained, indicated by preserved MitoTracker and mitochondrial immunostaining after PFA-GA fixation. Our results suggest that the PFA-GA combination is a valuable fixative for the study of mitochondria in situ.
Topics: Animals; Fixatives; Formaldehyde; Glutaral; Mice; Microscopy, Confocal; Mitochondria; Polymers
PubMed: 34068806
DOI: 10.3390/biom11050711