Madison Grant, Steven Foltz, Halie Zastre and Junna Luan provided technical assistance. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number AR065077. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

<ol>
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V., Asamoah, B., Gonzalez-Escalona, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+commercial+RNA+extraction+kits+for+preparation+of+DNA-free+total+RNA+from+Salmonella+cells.">Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells.</a> BMC Res. Notes. 3, 211 (2010).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> J. Vis. Exp. (45), e2565 (2010).</li><li>Valdez, M. R., Richardson, J. A., Klein, W. H., Olson, E. 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Pathol. 162 (5), 1685-1691 (2003).</li><li>Chomczynski, P., Mackey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+chloroform+by+bromo-chloropropane+in+the+single-step+method+of+RNA+isolation.">Substitution of chloroform by bromo-chloropropane in the single-step method of RNA isolation.</a> Anal. Biochem. 225 (1), 163-164 (1995).</li><li>Lee, J. T. Y., Cheung, K. M. C., Leung, V. Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+RNA+from+tough+tissues+with+high+proteoglycan+content+by+cryosection,+second+phase+separation+and+high+salt+precipitation.">Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation.</a> J. Biol. Methods. 2 (2), 20 (2015).</li><li>Guo, D., Catchpoole, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+RNA+following+cryosections+of+archived+frozen+tissue.">Isolation of intact RNA following cryosections of archived frozen tissue.</a> BioTechniques. 34, 48-50 (2003).</li><li>Goodpaster, T., Randolph-Habecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flexible+mouse-on-mouse+immunohistochemical+staining+technique+adaptable+to+biotin-free+reagents,+immunofluorescence,+and+multiple+antibody+staining.">A flexible mouse-on-mouse immunohistochemical staining technique adaptable to biotin-free reagents, immunofluorescence, and multiple antibody staining.</a> J. Histochem. Cytochem. 62 (3), 197-204 (2014).</li><li>Ikezawa, M., Minami, N., Takahashi, M., Goto, Y., Miike, T., Nonaka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+gene+analysis+of+130+patients+with+Duchenne+muscular+dystrophy+with+a+special+reference+to+muscle+mRNA+analysis.">Dystrophin gene analysis of 130 patients with Duchenne muscular dystrophy with a special reference to muscle mRNA analysis.</a> Brain Dev. 20 (3), 165-168 (1998).</li><li>Majumdar, G., Vera, S., Elam, M. B., Raghow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+streamlined+protocol+for+extracting+RNA+and+genomic+DNA+from+archived+human+blood+and+muscle.">A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.</a> Anal. Biochem. 474, 25-27 (2015).</li><li>Espina, V., Heiby, M., Pieroban, M., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+technology.">Laser capture microdissection technology.</a> Expert Rev Mol Diagnostics. 7 (5), 647-657 (2007).</li></ol>

The author declares that she has no competing financial interests.

Um die besten Ergebnisse mit dieser Methode zu erreichen, halten Harz auf das untere Drittel beschränkt Einbetten oder die Hälfte des Muskels während der Kryokonservierung Gewebe, weil überschüssiges Harz wird die Sammlung der gepoolten Kryoabschnitte verlangsamen und erhöhen Harz Kontamination in der RNA-Isolierung eingebettet ist. Auch besondere Aufmerksamkeit während der Nadel Homogenisierung ist wichtig, die Ausbeute zu maximieren und die Wahrscheinlichkeit einer Verstopfung der Nadel zu minimieren. Das Proto...

Das Ziel dieser Technik ist es, mehrere experimentelle Analysen von verschiedenen Modalitäten, wie Histologie und Genexpression, zugänglich von einem einzigen kleinen Skelettmuskel Quelle Gewebe zu machen. Mikroskopieanwendungen sind die empfindlichsten Konservierungsverfahren abzutasten, die sorgfältig während der Kryokonservierung kontrolliert werden muss, die Bildung von Eiskristallen Artefakte zu begrenzen. Somit wird der Methodenentwicklung auf der Grundlage der tibialis anterior (TA) gefroren Muskel teilweise mit Einbettungsharz in einer -140 ° C mit flüssigem Stickstoff gekühlt 2-methylbutan-Bad als Ausgangsmaterial sowohl für die Mikroskopie Immunofluoreszenz abgedeckt und Genexpressionsanalyse. Die Notwendigkeit, die gleiche Ausgangsmaterial für verschiedene technische Ansätze zu verwenden, ist besonders wichtig für die intramuskuläre Injektion basierende Experimenten, wo die linke und rechte Muskeln verschiedenen Bedingungen darstellen, eine Versuchs- und eine Kontroll. Zum Beispiel in der Muskelregeneration Studien, eine muscle ist mit einem Toxin injiziert weit verbreitete Gewebeschädigung zu verursachen , während der kontralateralen Muskel dient als Vehikel-injizierten Kontroll 1. In ähnlicher Weise Störungen Studien der Gentherapie für die Muskel beginnen in der Regel mit Validierung des Gentherapievektor durch intramuskuläre Injektion mit leerem Vektor verglichen werden, nicht verwandten Vektor oder Fahrzeugsteuerung auf der Gegenseite 2. Daher ist es nicht möglich, jeden TA Muskel zu einer anderen Anwendung beziehen. Gemeinsame Strategien mit diesem Thema befassen, sind: i) eine andere Muskelgruppe für jede Anwendung zu verwenden, ii) zusätzliche Mäuse zu verwenden, oder iii) für jede Anwendung ein Stück des Muskels abzuschneiden. Allerdings erhebliche Unterschiede zwischen den Muskelgruppen machen es schwierig, Daten aus separaten Anwendungen zu vergleichen und weiteren Tieren erhöhen Kosten und sind schlecht gerechtfertigt, wenn andere Alternativen gibt. Die Aufteilung der Muskel nach der Sektion verschiedenen Anwendungen zu beziehen ist die beste Option in vielen Fällen. Jedoch sind die Muskelstücke oft zu klein für die Homogenisierung 2-5 Pulverisierung unter flüssigem Stickstoff oder mechanische Schleiftechniken zu verwenden. Muskel führt ein hochStrukturGewebe gepackt mit extrazellulären Matrix und kontraktilen Proteine, unzureichende mechanische Homogenisierung zu einer geringen Ausbeute an nachfolgende DNA, RNA oder Protein. Das Verfahren detailliert hier können kleine Mengen von Gewebe aus einer Hand Muskel für den Einsatz in vielfältigen Anwendungen, und die Einbeziehung von Kryoschneiden und Nadel Verreiben verbessert mechanische Homogenisierung für eine bessere RNA-Ausbeute.

<table><tbody><tr><td>Cork</td><td>VWR Scientific</td><td>23420-708</td><td>Cut into small squares with a sharp blade.</td></tr><tr><td>Plastic coverslip</td><td>Fisher Scientific</td><td>12-547</td><td>Used to orient the muscle during freezing.</td></tr><tr><td>Low temperature thermometer</td><td>VWR Scientific</td><td>89370-158</td><td></td></tr><tr><td>2-methylbutane</td><td>Sigma</td><td>M32631-4L</td><td>Caution: hazardous chemical. Store in flammable cabinet.</td></tr><tr><td>Embedding resin: &quot;cryomatrix&quot;</td><td>Thermo Fisher Scientific</td><td>6769006</td><td>Other embedding resins can be substituted for cryomatrix.</td></tr><tr><td>Cryostat</td><td>Thermo Fisher Scientific</td><td>microm HM550 with disposable blade carrier</td><td>Any working cryostat should be sufficient for the protocol.</td></tr><tr><td>Disposable cryostat blade</td><td>Thermo Fisher Scientific</td><td>3052835</td><td>Use an appropriate blade or knife for the cryostat to be used.</td></tr><tr><td>RNAse decontamination solution: &quot;RNase Zap&quot;</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>XS64</td><td></td></tr><tr><td>Paint brush</td><td>Daler Rowney</td><td>214900920</td><td>Use to handle cryosections. Can be found with in stores with simple art supplies.</td></tr><tr><td>Razor blade</td><td>VWR Scientific</td><td>55411-050</td><td></td></tr><tr><td>Microscope slide</td><td>VWR Scientific</td><td>48311600</td><td></td></tr><tr><td>RNA organic extraction reagent: TRIzol</td><td>Thermo Fisher Scientific</td><td>15596026</td><td>Caution: TRIzol is a hazardous chemical. Note: Only organic extraction reagents are recommended for RNA extraction from skeletal muscle.</td></tr><tr><td>18 gauge needle</td><td>VWR Scientific</td><td>BD305185</td><td></td></tr><tr><td>22 gauge needle</td><td>VWR Scientific</td><td>BD305155</td><td></td></tr><tr><td>26 gauge needle</td><td>VWR Scientific</td><td>BD305115</td><td>Optional. Can be used for a third round of sample trituration in the RNA extraction protocol.</td></tr><tr><td>1 ml syringe</td><td>VWR Scientific</td><td>BD309659</td><td>For very high value samples, a Luer-Lok syringe is recommended (&lt;i&gt;e.g.&lt;/i&gt;, VWR BD309628).</td></tr><tr><td>1-bromo-3-chloropentane (BCP)</td><td>Sigma</td><td>B9673</td><td></td></tr><tr><td>For 70% ethanol in DEPC water: 200 proof alcohol</td><td>Decon Laboratories, Inc.</td><td>+M18027161M</td><td>Mix 35 ml 200 proof alcohol + 15 ml DEPC water.&amp;nbsp;</td></tr><tr><td>For 70% ethanol in DEPC water: DEPC-treated water</td><td>Thermo Fisher Scientific</td><td>AM9922</td><td>Mix 35 ml 200 proof alcohol + 15 ml DEPC water.</td></tr><tr><td>RNA purification kit: PureLink RNA minikit</td><td>Thermo Fisher Scientific</td><td>12183018A</td><td>Final steps of RNA preparation.</td></tr><tr><td>DNase/Rnase-free water&amp;nbsp;</td><td>Gibco</td><td>10977</td><td>DEPC-treated water can also be used.</td></tr><tr><td>Spectrophotometer: Nanodrop 2000</td><td>Thermo Fisher Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Dnase I</td><td>Thermo Fisher Scientific</td><td>AM2222</td><td>Treat purified RNA to remove any DNA contamination before downstream appications.</td></tr><tr><td>Hydrophobic pen</td><td>Thermo Fisher Scientific</td><td>8899</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Gibco</td><td>14190</td><td>PBS for immunofluorescence protocol.</td></tr><tr><td>Donkey serum</td><td>Jackson ImmunoResearch Laoratories, Inc</td><td>017-000-121</td><td>Rehydrate normal donkey serum stock according to the manufacturer&#039;s instructions, then dilute an aliquot to 5% for immunofluorescence.&amp;nbsp; Normal goat serum can also be used.</td></tr><tr><td>eMHC antibody</td><td>University of Iowa Developmental Studies Hybridoma Bank</td><td>F1.652</td><td></td></tr><tr><td>Collagen VI antibody</td><td>Fitzgerald Industries</td><td>#70R-CR009x</td><td></td></tr><tr><td>Donkey anti-rabbit AlexaFluor488</td><td>Thermo Fisher Scientific</td><td>A21206</td><td></td></tr><tr><td>Goat anti-mouse IgG1 AlexaFluor546</td><td>Thermo Fisher Scientific</td><td>A21123</td><td></td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Aqueous mounting media: Permafluor</td><td>Thermo Fisher Scientific</td><td>TA-030-FM</td><td>Only use mounting media designed for fluorescent applications with anti-fade properties.</td></tr><tr><td>Glass coverslip</td><td>VWR Scientific</td><td>16004-314</td><td>Use for mounting slides at the end of immunofluorescence protocl</td></tr><tr><td>Image analysis software: ImageProExpress</td><td>Media Cybernetics, Inc.</td><td>Image-Pro Express, or more advanced products</td><td>Freeware ImageJ should also work for manual counting. More advanced software with segmentation abiities may allow partial automation of the process; &lt;em&gt;e.g.&lt;/em&gt;, ImageProPremier.</td></tr><tr><td>Merge and map section images: Photoshop</td><td>Adobe</td><td>Photoshop</td><td></td></tr><tr><td>Cardiotoxin</td><td>Sigma</td><td>C9659</td><td>Sigma C9659 has been discontinued. Other options for cardiotoxin are EMD Millipore #217503; American Custom Chemicals Corp. # BIO0000618; or Ge Script # RP17303; but these have not been validated.</td></tr><tr><td>reverse transcription kit: Superscript III First-strand synthesis system</td><td>Thermo Fisher Scientific</td><td>18080051</td><td>Any validated, high quality reverse transcription reagents can be used.</td></tr><tr><td>Standard PCR: GoTaq Flexi polymerase system</td><td>Promega</td><td>M8298</td><td>Any validated, high quality Taq polymerase system can be used. If DNA sequencing is to be used for any application downstream of the PCR, then a high fidelity PCR system should be used instead.</td></tr><tr><td>SYBR green</td><td>Thermo Fisher Scientific</td><td>S7585</td><td>For use in qPCR when not using a dedicated qPCR master mix. Use with SuperROX (for Applied Biosystems instruments) and GoTaq Flexi polymerase and buffers.</td></tr><tr><td>ROX: SuperROX, 15 mM</td><td>BioResearch Technologies, Inc. Novato CA</td><td>SR-1000-10</td><td>SuperROX is more stable in the PCR reaction, so it is preferred for use as a qPCR passive reference dye over ROX (carboxy-X-rhodamine). For qPCR with Applied Biosystems instruments</td></tr><tr><td>Real-time PCR</td><td>Applied Biosystems</td><td>7900HT</td><td></td></tr></tbody></table>

cryosectioning of contiguous regions of a single mouse skeletal muscle for gene expression and histological analyses

Mit diesem Verfahren werden aufeinanderfolgende Kryoschnitte gesammelt beide Mikroskopieanwendungen für Gewebe Histologie und Anreicherung von RNA für die Genexpression zu ermöglichen, benachbarte Bereiche von einer einzigen Maus Skelettmuskel verwendet. Typischerweise wird es ausreichende Homogenisierung von kleinen Skelettmuskelproben zu erreichen, herausfordernd, weil Puffervolumen für die effiziente Schleifanwendungen zu gering sein kann, aber ohne ausreichende mechanische Störung, die dichte Gewebearchitektur der Muskelgrenzen Eindringen von Pufferreagenzien, was letztlich geringe RNA-Ausbeute. Durch Befolgen des Protokolls hier berichtet wird, 30 &amp; mgr; m Abschnitte werden gesammelt und vereinigt Kryoschneiden und anschließende Homogenisierung Nadel ermöglicht, um mechanisch den Muskel zu stören, die Erhöhung der Oberfläche für die Puffer Eindringen ausgesetzt. Die primären Beschränkungen der Technik sind, dass es einen Kryostaten erfordert, und es ist relativ geringen Durchsatz. Allerdings können qualitativ hochwertige RNA aus kleinen Proben von gepoolten m erhalten werdenuscle Kryoschnitte, so dass dieses Verfahren zugänglich für viele verschiedene Skelettmuskeln und anderen Geweben. Darüber hinaus ermöglicht diese Technik angepasst Analysen (zB Gewebe Histopathologie und Genexpression) aus benachbarten Regionen eines einzelnen Skelettmuskel , so dass Messungen direkt im Vergleich über mehrere Anwendungen hinweg werden können experimentelle Unsicherheit zu reduzieren und replikative Tierversuche notwendig zur Quelle ein kleines Gewebe zu reduzieren für mehrere Anwendungen.

Muscle Kryoschnittes RNA ist hoch in der Qualität und sorgt für eine ausreichende Ausbeute für die meisten Anwendungen Die Analysen von sechzehn Skelettmuskel - RNA - Präparationen sind in Tabelle 1 unter Verwendung von 19,4 bis 41 mg gepoolten tibialis anterior (TA) Muskel von 8 Kontrollmäusen gezeigt. Sowohl linke (L) und rechten (R) TA Muskeln wurden in Regenerationsversuche vorbereitet mit 3 Tagen gesamm...

Watch this Scientific Journal Video about Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses at JoVE.com

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

Aufeinanderfolgende Kryo-Abschnitte werden gesammelt, um histologische Anwendungen und Anreicherung von RNA für Genexpressionsmessungen mit benachbarten Regionen aus einer einzigen Maus Skelettmuskel ermöglichen. 30 mg gepoolt Kryoabschnitte und Messungen werden direkt im Vergleich über mehrere Anwendungen hinweg - Qualitativ hochwertige RNA wird aus 20 erhalten.

Kryoschneiden von benachbarten Regionen eines einzelnen Maus Skelettmuskel für die Genexpression und histologische Analysen

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

cryosectioning-contiguous-regions-single-mouse-skeletal-muscle-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.6K Aufrufe.  University of Georgia.  Aufeinanderfolgende Kryo-Abschnitte werden gesammelt, um histologische Anwendungen und Anreicherung von RNA für Genexpressionsmessungen mit benachbarten Regionen aus einer einzigen Maus Skelettmuskel ermöglichen. 30 mg gepoolt Kryoabschnitte und Messungen werden direkt im Vergleich über mehrere Anwendungen hinweg - Qualitativ hochwertige RNA wird aus 20 erhalten. 

Serie: Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Non-Laser Capture Microscopy Ansatz für die Mikrodissektion von Discrete-Maus Hirnregionen für Gesamt-RNA Isolierung und Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Forschung

Neurowissenschaften

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser Capture Mikrodissektion von Maus embryonalen Knorpel und Knochen für Die Genexpressionsanalyse

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Entwicklungsbiologie

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

Der neuromuskulären Synapse: Mess Synapse Größe, Fragmentierung und Veränderungen der synaptischen Protein Dichte Mit der konfokalen Fluoreszenzmikroskopie

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Gebote und Verbote in der Herstellung von Muscle Kryoschnitte für die histologische Analyse

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

Medizin

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Gleichzeitige Kryoschneiden mehrere Nagetier Gehirne

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

Mikrodissektion des Mausgehirns in funktionell und anatomisch unterschiedliche Regionen

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Kryoschneiden Verfahren zur Mikrodissektion von Murine Kolonschleimhaut

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Biologie

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Fasertyp- und subzellspezifische Analyse des Lipidtröpfchengehalts in der Skelettmuskulatur

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

Ein Laser Capture Microdissection Protocol, das hochwertige RNA aus frisch gefrorenen Mausknochen liefert

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Enzymatische Isolierung und Charakterisierung von Mausmuskelfasern variabler Länge

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Autor im Rampenlicht: Isolierung langer Muskelfasern aus den Hintergliedmaßenmuskeln von Mäusen zur Untersuchung der Erregungs-Kontraktions-Kopplung zwischen Fasertypen

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Intakte kurze, mittlere und lange Skelettmuskelfasern, die durch enzymatische Dissoziation von sechs Hintergliedmaßenmuskeln von Mäusen gewonnen wurden: Jenseits des Flexor Digitorum brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...