Name: the cutting and floating method for paraffin-embedded tissue for sectioning
Uploaded: 2018-09-05T15:30:00
Description: Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

Dette arbeidet ble støttet av National Natural Science Foundation of China (Grant nr. 31400936, 31460260) og den Natural Science Foundation i Jiangxi-provinsen i Kina (20171BAB215020). Vi takker også det felles programmet mellom Nanchang University og Queen Mary University of London for å støtte dette arbeidet.

Alle metodene som er beskrevet her er godkjent av Animal Care og bruk komiteen av Nanchang University.
1. samle utstyret og koble mikrotomen
<ol><li>Utforme parameteren per kravene (utfyllende figur 1).</li>
	<li>Sende parameteren til en lokal fabrikk å produsere akryl styrene.</li>
	<li>Samle alle deler i rekkefølge: bruke kloroform kombinere 7 kommersielle akryl styrene i en tank med delen kanal og vann bad (figur 1</str...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+zebrafish+histology.">High-throughput zebrafish histology.</a> Methods. 39 (3), 246-254 (2006).</li><li>Chen, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid-Cut:+An+Improved+Sectioning+Method+for+Recalcitrant+Plant+Tissue+Samples.">Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples.</a> Journal of Visualized Experiments. (117), e54754 (2016).</li><li>Kucherenko, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin-Embedded+and+Frozen+Sections+of+Drosophila+Adult+Muscles.">Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles.</a> Journal of Visualized Experiments. (46), e2438 (2010).</li><li>Cornell, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+and+Thin+Sectioning+of+Microbial+Colony+Biofilms+for+Microscopic+Analysis.">Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis.</a> Journal of Visualized Experiments. (133), e57196 (2018).</li><li>Whiteland, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+detection+of+T-cell+subsets+and+other+leukocytes+in+paraffin-embedded+rat+and+mouse+tissues+with+monoclonal+antibodies.">Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.</a> Journal of Histochemistry and Cytochemistry. 43 (3), 313-320 (1995).</li><li>Tucker, D. K., Foley, J. 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For å forbedre parafin delen morfologi og løse problemet med bortkastet tid under konvensjonell parafin snitting, laget vi en forbedret parafin snitting metoden som kombinerer skjæring og unfolding. Denne forbedrede metoden er avhengig av enkel utstyr som består av en delen kanal, et vannbad og en oppvarmet med temperatur oppdagelsen bryter. Delen båndet inn vannbad gjennom delen kanalen og utfolder seg automatisk under sagingen. Derfor forbedrer denne metoden parafin snitting kvalitet og effektivitet, som ble bekre...

Morfologisk studie er viktig i biologisk forskning. Selv om ny teknologi har tillatt forskere å observere sine mål direkte fra hele vev eller organismer1,2,3, kutte prøven i tynne snitt, etterfulgt av flekker, forblir primært metoden fornot eneste vev morfologi, men også protein målretting direkte i vevet. * Lys bruker tre inndelingstypene: parafin, frossen og semithin. Selv om og kryosnitt er vanlig for å beskytte vev antigenicity og prøven forberedelsene er enkel, er beholdt vev morfologi fattige og uegnet for tynne snitt4,5. Parafin snitting er den mest brukte metoden for utstilling godt bevart morfologi. De er dehydrert helt og innebygd i voks, kan parafinblokkene lagres på ubestemt tid. I tillegg produserer parafin snitting tynne snitt som bedre biologiske sonde tilgang i videre studier og redusere celle lag overlegg i Z-retningen.
Men konvensjonell parafin snitting er kjedelig og krever operatør ferdigheter. Parafinsnitt gjennomgå fiksering, dehydrering, innebygging, skjæring og flytende. Viktigere, er overføre delen bånd fra knivholderen til vannbad nødvendige, men vanskelig for junior operatører. Spesielt i tørr luft, delen bånd vil vri på grunn av statisk elektrisitet og er vanskelig å utfolde seg på varmt vannoverflaten. For å forbedre delen anbefales kvalitet, fukte eksponert vevet overflaten mellom mikrotomen blad passerer, kjøling voks blokkene ved å leve dem i isvann, eller høyne fuktigheten med en luftfukter nær mikrotomen6,7 . Nyere metoder for å forbedre parafin snitting inkluderer hybrid parafin innebygging og kryosnitt8og kommersielle delen overføring system hjelp9. Selv om disse metodene delvis forbedre parafin snitting hastighet og kvalitet, de gjør snitting mye mer tungvint, og transportsystemer for kommersielle delen er dyrt.
Denne protokollen viser vi hvordan å lage enkle, billige og fleksibel utstyr steg for steg, som kan være koblet til holderen over en roterende mikrotom. Utstyret består av en delen kanal, et vannbad og en oppvarmet med temperatur oppdagelsen bryter. Etter klipping, dusinvis av deler strømme inn delen kanalen og angi vannbad på direkte, dermed utspiller seg automatisk. Dette forbedrer effektiviteten av parafin snitting og gjør denne teknologien mer praktisk. Bruker denne metoden, mer voksen mus hippocampus deler, voksen mus nyre deler, embryonale 15.5 dager gamle (E15.5) musen hjernen inndelinger og voksen sebrafisk øye delene ble høstet i mindre tid og forble mer intakt morphologically. Denne metoden kan også brukes for andre vevsprøver som krever akselerert parafin snitting og unngå tap av delen skillet.

<table>
	<tbody>
		<tr>
			<td>Incubator</td>
			<td>Boekel Scientific</td>
			<td>133000-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Leica</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Paraffin Embedding Module</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial acrylic board</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubular electric heating element(12V 200W)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller(12v 120w)</td>
			<td>Mingsuo</td>
			<td>XH-W3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary microtome&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral silicone sealant</td>
			<td></td>
			<td></td>
			<td>Link the water channel with the microtome knife holder</td>
		</tr>
		<tr>
			<td>Voltage transformer</td>
			<td>Dearll</td>
			<td>S-250-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable blade</td>
			<td>Accu-Edge</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Baso Diagnostics Inc.</td>
			<td>BA-4025</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Baso Diagnostics Inc.</td>
			<td>BA-4025</td>
			<td></td>
		</tr>
		<tr>
			<td>Microslide</td>
			<td>Sail Brand</td>
			<td>7105</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>10004160</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip&#160;</td>
			<td>Citoglas</td>
			<td>10212424C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Xilong Chemical</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath for paraffin sections</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HistoCore Arcadia C&#160;- Cold Plate</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>paraffin repellent spray&#160;</td>
			<td>Thermo Scientific</td>
			<td>9990420</td>
			<td></td>
		</tr>
	</tbody>
</table>

the cutting and floating method for paraffin-embedded tissue for sectioning

Skjæring av parafin-embedded vev er mye brukt i histologi og patologi. Men er det kjedelig. For å forbedre denne metoden, har flere kommersielle selskaper utarbeidet komplekse delen transportsystemer med flytende vann. For å forenkle denne teknologien, laget vi en enkel metode bruker hjemmelaget utstyr som kombinerer kutte og flytende innen en enkelt Termostatiske kammer; Derfor inn delene automatisk vannbad på vannflaten. Hippocampus fra voksen mus hjerner, voksen mus nyrer, embryonale musen hjerner og voksen sebrafisk øyne ble skåret med både konvensjonell parafin snitting og presentert metoden for sammenligning. Statistiske analyser viser at vår forbedret metode lagret tid og produsert høyere kvalitet deler. I tillegg er parafin snitting av en hel på kort tid lett for junior operatører.

Forbedret metoden økt antall intakt parafinsnitt. Vi testet denne nye metoden på voksen mus hippocampus vev, voksen mus nyrer, embryonale musen hjerner og sebrafisk øyne. Vannet ble lagt til tanken, og temperaturen ble opprettholdt 38.0 ° c til 40,0 ° C. Etter et serielt forbereder vevsprøver, var de delt og sammenlignet med vanlig snittemetode. Den nye metoden unngått delen tap og økt andelen intakt deler i voksen mus hippocampus, voksen mus nyrene, E15.5 musen hjerner og voksen ...

Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Skjæring og flytende metode for parafin-embedded vev snitting

Her presenterer vi en protokoll for å forbedre parafin snitting. Denne metoden kombinerer kutte og flytende bruker en enkel Termostatiske kammer for å unngå overføringsprosessen kreves av det tradisjonelle metoden. Som et resultat, ble effektiviteten og antall intakt parafinsnitt kraftig forbedret.

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

This method can help answer key questions in morphological field, such as paraffin sectioning. The main advantage of this technique is that culminates cutting and the folding procedure and avoid transfer process that required by conventional method. To begin, gather all of the pre-manufactured parts in a fume hood.Use chloroform to combine the seven acrylic boards, as shown here. The end product will be a tank equipped with a section channel and a water bath. Install the tubular electrical heating element through the hole in acrylic board number two.Using an electric drill, make a small hole in acrylic board number seven, and install a digital thermostat on the tank, making sure that it is at least one centimeter below the level of the intended water surface, which is indicated by the black dot on the tank. Adjust the voltage so that it is below 24 volts before turning on the equipment. Remove the section waste tray from the microtome.Next, use neutral silicone sealant to link the sections channel to the microtome blade holder, making sure not to get any sealant above the top of the blade. Let the setup dry overnight to give the chloroform and neutral silicone sealant time to solidify. The next day, install the blade in the blade holder.Add water to the water bath, making sure that the water surface just touches the top of the blade. After performing conventional paraffin sectioning, turn on the equipment to begin improved paraffin sectioning. Use the temperature detection switch to open the heater.Set the targeted temperature between 38 degrees Celsius and 40 degrees Celsius for one hour to warm the water. Use a knife to modify the paraffin block acquired by paraffin embedding. Then, place the block on the microtome's cassette clamp, and lock it.Cut the thicker sections to quickly approach the sample. Adjust to a suitable section thickness for the samples. Turning the hand wheel will automatically load the sections into sections channel and bath.Next, section the specimen in a slow and uniform cutting stroke. Guide the sections into the bath chamber so that they float in a line. If the head section adheres to the chamber wall, use a brush guide to keep it floating.After the sections fully unfold in the water bath, use tweezers to separate several, and then pick them up on a microscope slide. Transfer the slides to an oven set to 42 degrees Celsius to dry overnight. The slides can then be stored at room temperature indefinitely until ready to analyze.When ready to analyze, use hematoxylin-eosin staining to evaluate the sections using the improved method. In this study, an improved method is used to prepare paraffin sections for adult mouse hippocampal tissue, adult mouse kidneys, embryonic mouse brains, and zebrafish eyes. As shown here, the new method avoids section loss and increases the proportion of intact sections in each type of examined tissue.When performing whole-series sectioning, this method is seen to accelerate the paraffin sectioning speed in all tested samples. In other words, this method provides higher quality paraffin sectioning in less time than the conventional method. Series sections of zebrafish optic retina are then observed by hematoxylin-eosin staining.The sections harvested from the improved method are seen to have better integrity than those harvested from the conventional paraffin sectioning method. While there are no significant differences in the quality of most harvested sections when comparing these two methods, some sections harvested from the conventional method are seen to have relatively low quality. Don't forget that working with chloroform can be extremely hazardous, and precautions such as fume hood should always be taken while performing this procedure.While attempting this procedure, it's important to remember, add water carefully and avoid overfill.

Research

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Neuroscience

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

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Immunhistokjemisk analyse i Rat sentralnervesystemet og perifer lymfeknute vevsdelene

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

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Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Utarbeidelse av akutt hjernen skiver med en optimalisert N-Methyl-D-glucamine beskyttende gjenopprettingsmetode

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

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Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

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Langvarig Inkubasjon av akutt nervevev for Elektro og kalsium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

Målretting Alpha Synuclein aggregater i hud perifere nerve fibre av fri-flytende Immunofluorescence analysen

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Kortsiktige fritt flytende Slice kulturer fra Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

En hydrofob vevsryddingsmetode for rottehjernevev

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

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Seriell blokk-ansiktsskanning elektronmikroskopi (SBEM) for studiet av dendritiske spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...