Name: correlative light and electron microscopy to study microglial interactions with &#946;-amyloid plaques
Uploaded: 2016-06-01T15:00:00
Description: Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃŽÂ²-Amyloid Plaques at JoVE.com

We are grateful to Dr. Sachiko Sato and Julie-Christine L&#233;vesque at the Bioimaging Platform of the Centre de recherche du CHU de Qu&#233;bec for their technical assistance. Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2014-05308, The Banting Research Foundation, and The Scottish Rite Charitable Foundation of Canada to M.E.T supported this work.
H.E.H. is recipient of a scholarship from the Lebanese Ministry of Education and Higher Education, and K.B. from the Facult&#233; de m&#233;decine of Universit&#233; Laval.

Bemærk: Alle forsøg blev godkendt og udført i henhold til retningslinjerne i den institutionelle dyreetik udvalg, i overensstemmelse med den canadiske Rådet om Animal Care retningslinjer som administreres af Animal Care udvalg Université Laval. APP-PS1 hanmus mellem 4 og 21 måneder gamle, blev anvendt. Disse dyr blev huset under en 12 h lys-mørke-cyklus ved 22 - 25 ° C med fri adgang til foder og vand. 1. Methoxy-x 04 Fremstilling af opløsning <ol><li> Der fremstilles en 5 mg / ml...

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Denne protokol beskrives en korrelative tilgang til målretning tæt kerne Ap plaques med EM. Methoxy-x 04 in vivo injektion muliggør hurtig udvælgelse af hjernen sektioner, der indeholder Ap plaques inden for bestemte regioner og lag af interesse, for eksempel hippocampus CA1, strata radiatum, og lacunosum-moleculare. I det foreliggende eksempel blev methoxy-x 04 pre-screening kombineret med præ-indlejring immunfarvning for IBA1 at studere, hvordan forskellige microgliale fænotyper interagere med synapser ...

Amyloid Ap plakdannelse er det vigtigste neuropatologiske kendetegnende for Alzheimers sygdom (AD). Men stigende tyder vigtige roller i immunsystemet i sygdomsprogression 1,2. Især nye data fra prækliniske og kliniske studier etableret immun dysfunktion som en vigtigste drivkraft og bidragyder til AD patologi. Med disse resultater, har centrale og perifere immunceller dukket op som lovende terapeutiske mål for AD 3. Den følgende protokol kombinerer lys og elektronmikroskopi (EM) til at generere nye indsigter i forholdet mellem Ap plaque deposition og microgliale fænotypiske ændringer i AD. Denne protokol tillader mærkning af Ap plaques i musemodeller for AD under anvendelse af in vivo injektion af det fluorescerende farvestof methoxy-x 04. Methoxy-x 04 er en Congo Red-derivat, der let kan krydse blod-hjerne-barrieren til at indtaste hjerneparenkymet og binde β-plisserede ark med højffinity. Eftersom farvestoffet er fluorescerende, kan den anvendes til in vivo detektion af Ap plaque deposition med to-foton mikroskopi 4. Når bundet til Ap, betyder methoxy-x 04 ikke tager afstand eller videredistribuere væk fra plaques, og det bevarer sin fluorescens over tid. Det er generelt indgives perifert at muliggøre ikke-invasiv billeddannelse af hjernen dynamik 5. Fluorescensen forbliver også efter aldehyd fiksering, der giver mulighed for korrelativ post mortem-analyser, herunder undersøgelse af neuronal død i nærheden af Ap-plaques 6. Denne protokol udnytter egenskaberne af methoxy-x 04 for at vælge hjerne sektioner fra APP SWE / PS1A 246e mus (APP-PS1, co-udtrykke en dobbelt mutation på APP genet Lys670Asn / Met671Leu, og human presenilin PS1-A264E variant) 7, der udviser Ap plaques i specifikke områder af interesse (hippocampus CA1, strata radiatum og lacunosum-moleculare) Før pre-embedding immunfarvning mod mikroglial markør ioniseret calcium-bindende adapter molekyle 1 (IBA1) at visualisere microgliale celle organer og processer med EM. Musene gives intraperitoneal injektion af methoxy-x 04-opløsning, 24 timer før hjernen fiksering gennem transcardial perfusion. Koronale hjernesnit opnås ved anvendelse af et vibratom. Sektioner indeholdende hippocampus CA1 screenes under et fluorescerende mikroskop for tilstedeværelse af Ap plaques i strata radiatum og lacunosum-moleculare. Immunfarvning for IBA1, er osmiumtetroxid post-fiksering, og plast harpiks indlejring derefter udført på de udvalgte hjernesektioner. Ved slutningen af ​​denne protokol, kan sektionerne arkiveres uden yderligere ultrastrukturelle nedbrydning, klar til ultratynde sektionering og ultrastrukturelle undersøgelse. Vigtigere er de plaques stadig fluorescerende efter immunfarvning med forskellige antistoffer, f.eks IBA1 som i den foreliggende protokol. De bliver mørkere tHan deres omgivende neuropil efter osmiumtetroxid post-fiksering, uafhængigt af methoxy-x 04 farvning, som hjælper med at præcist at identificere de områder af interesse, generelt ned til nogle få kvadratmillimeter, der skal undersøges med transmissions elektron mikroskop. Denne korrelative tilgang giver en effektiv måde at identificere specifikke hjerne sektioner for at undersøge på ultrastrukturelle niveau. Dette er især nyttigt, når studerer tidlig AD patologi, inden specifikke hjerneregioner eller lag, som kun indeholder nogle få Ap plaques, der er til stede i kun en lille brøkdel af vævssnit. I disse tider især, ville det være effektivt at anvende immunfarvning for Ap (og dobbelt mærkning for andre cellulære markører, såsom IBA1) på flere hjernesnit blot for at give en lille fraktion indeholdende Ap plaques på det rigtige sted. Desuden injektion af levende mus med methoxy-x 04 før aflivning og væv behandlingen ikke compromise den ultrastrukturelle konservering. Alternative metoder såsom farvning post mortem med Congo Rød, Thioflavin S, thioflavin T eller methoxy-x 04 på faste sektioner væv kræver farvning differentiering i ethanol, 8-11, som forårsager osmotiske stress og forstyrrer ultrastruktur. Congo Red er også kræftfremkaldende for mennesker 12.

<table border="0" cellpadding="0" cellspacing="0" width="1006">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Methoxy-X04</td>
			<td style="width:197px;">Tocris Bioscience</td>
			<td style="width:119px;">4920</td>
			<td style="width:167px;">10 mg substrate per tablet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propylene glycol</td>
			<td>Sigma Aldrich</td>
			<td>W294004&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher BioReagents</td>
			<td>BP231-1</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Hydrochloride</td>
			<td>Fisher BioReagents</td>
			<td>BP153500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrolein&#160;</td>
			<td>Sigma</td>
			<td>110221</td>
			<td>Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde Granular</td>
			<td>Electron Microscopy Sciences</td>
			<td>19210</td>
			<td>Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper</td>
			<td>Fisher</td>
			<td>09-790-14F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump with Tubing</td>
			<td>ColeParmer</td>
			<td>cp.78023-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel Winged blood Collection Set Needles 25G&#160;</td>
			<td>Becton Dickinson</td>
			<td>367341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extrafine Forceps</td>
			<td>F.S.T</td>
			<td>11152-10</td>
			<td>Tip shape: curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors</td>
			<td>F.S.T</td>
			<td>14090-09</td>
			<td>Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td>F.S.T</td>
			<td>13003-10</td>
			<td>Tip shape: curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors</td>
			<td>F.S.T</td>
			<td>14004-16</td>
			<td>Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Dissecting Scissors</td>
			<td>ROBOZ surgical store</td>
			<td>5818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>74515-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibrating Blade Michrotome Leica VT1000 S</td>
			<td>Leica Biosystems&#160;</td>
			<td>14047235612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome blades</td>
			<td>Electron microscopy Sciences</td>
			<td>71990</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Tissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene Glycol</td>
			<td>Fisher BioReagents</td>
			<td>BP230-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen Peroxide, 30%</td>
			<td>J.T.BAKER</td>
			<td>cat: 2186-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium borohydride</td>
			<td>Sigma</td>
			<td>480886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl</td>
			<td>Fisher</td>
			<td>BP153-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine Serum (FBS)</td>
			<td>Sigma Aldrich</td>
			<td>F1051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA), fraction V</td>
			<td>Thomas Scientific</td>
			<td>C001H24</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti IBA1, Rabbit&#160;</td>
			<td>WAKO</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rabbit IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>111066046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASTAIN Elite ABC Kit (Standard)</td>
			<td>Vector Labs</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.3&#39;-Diaminobenzidine tetra-hydrochloride (DAB)</td>
			<td>Sigma</td>
			<td>D5905-50TAB</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osmium tetroxide, 4% solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>19150</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component A</td>
			<td>Sigma</td>
			<td>44611</td>
			<td>Resin Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component B</td>
			<td>Sigma</td>
			<td>44612</td>
			<td>Hardener Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component C</td>
			<td>Sigma</td>
			<td>44613</td>
			<td>Plasticizer Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component D</td>
			<td>Sigma</td>
			<td>44614</td>
			<td>Accelerator Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>LesAlcoolsdeComerce</td>
			<td>151-01-15N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propylene oxide</td>
			<td>Sigma</td>
			<td>110205</td>
			<td>Caution: corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum weigh dishes</td>
			<td>Electron Microscopy Sciences</td>
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			<td height="21" style="height:21px;">ACLAR&#174;&#8211;Fluoropolymer Films</td>
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			<td height="21" style="height:21px;">Oven/Incubator</td>
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correlative light and electron microscopy to study microglial interactions with &#946;-amyloid plaques

En detaljeret protokol er anført her for at identificere amyloid Ap plaques i hjernen sektioner af Alzheimers sygdom musemodeller før præ-embedding immunfarvning (specifikt for ioniseret calcium-bindende adaptormolekyle 1 (IBA1), et calcium-bindende protein udtrykt af mikroglia) og vævsbehandling for elektronmikroskopi (EM). Methoxy-x 04 er et fluorescerende farvestof, der krydser blod-hjerne-barrieren og selektivt binder til p-foldet ark findes i tæt kerne Ap plaques. Injektion af dyrene med methoxy-x 04 før aflivning og hjerne fiksering tillader pre-screening og udvælgelse af plaque-holdige hjerne sektioner for yderligere behandling med tidskrævende manipulationer. Dette er især nyttigt, når studerer tidlig AD patologi inden for specifikke områder af hjernen eller lag, der kan indeholde meget få plaques, der er til stede i kun en lille brøkdel af sektionerne. behandling af vævssnit med Congo Rød, Thioflavin S, og Thiofl Post-mortemavin T (eller endda med methoxy-x 04) kan mærke p-plisseret ark, men kræver omfattende rydning med ethanol for at fjerne overskydende farvestof og disse procedurer er uforenelige med ultrastrukturelle konservering. Det ville også være effektivt at udføre mærkning af Ap (og andre cellulære markører, såsom IBA1) på alle hjernesnit fra regioner af interesse, kun for at give en lille fraktion indeholdende Ap plaques på det rigtige sted. Vigtigere er det, Ap plaques er stadig synlige efter vævsbehandling for EM, der giver mulighed for en præcis identifikation af de områder (generelt ned til nogle få kvadratmillimeter) med elektronmikroskop til at undersøge.

Dette afsnit viser de resultater, der kan opnås ved forskellige kritiske trin i protokollen. Især resultaterne viser eksempler på hjernen sektioner indeholder methoxy-x 04 farvede plaques i bestemt region og lag af interesse: hippocampus CA1, strata radiatum og lacunosum-moleculare. De plaques og regionale / lamellar organisering af hippocampus successivt visualiseres ved hjælp af en kombination af UV og lyse felt filtre (Figur 1). De valgte hjernesnit efterfølgende...

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃŽÂ²-Amyloid Plaques at JoVE.com

Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃƒÅ½Ã‚Â²-Amyloid Plaques

I denne artikel beskrives en protokol til visualisering amyloid Ap plaques i Alzheimers sygdom-musemodeller ved anvendelse methoxy-x 04, som krydser blod-hjerne-barrieren og selektivt binder til p-plisserede ark findes i tæt kerne Ap plaques. Det tillader præ-screening af plaque-holdige vævssnit før immunfarvning og forarbejdning til elektronmikroskopi.

Korrelative lys- og elektronmikroskopi at studere microgliale Interaktioner med p-amyloid plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Research

JoVE Journal

Neuroscience

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light Preference Assay til at studere Medfødte og Circadian Reguleret Photobehavior i<em&gt; Drosophila</em&gt; Larver

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Micron-skala Opløsning Optical Tomography af hele Mouse Brains med konfokal Light Sheet Microscopy

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetisk manipulation af Cerebellær granuleneuroner<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; At studere Neuronal morfologi og Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

Det olfaktoriske system som en model til at studere axonal vækstmønstre og morfologi<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

En menneskelige blod-hjerne Interface Model til at studere barriere overfarter af patogener eller lægemidler og deres interaktioner med hjernen

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Let induceret Molekylær adsorption af proteiner ved hjælp af PRIMO-systemet til Mikromønster for at studere celle respons på ekstracellulære matrix proteiner

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Kegle-beriget kulturer fra nethinden af kyllingembryoner til at studere Rod til Cone Cellular Interaktioner

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

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

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

Seriel blok-ansigt scanning elektron mikroskopi (SBEM) til undersøgelse af dendritiske rygsøjler

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

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

Primære kulturer af rotteastrocytter og microglia og deres anvendelse i undersøgelsen af amyotrofisk lateral sklerose

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...