Name: immunohistochemical and calcium imaging methods in wholemount rat retina
Uploaded: 2014-10-13T13:00:00
Description: Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

Vi takker Dr. S. Stella og Helen Vuong for bidrag til calcium imaging-protokollen. Vi takker Dr. K. Ark til at filme interviewet scener. Vi takker Dr. Arlene Hirano for hendes kommentarer til manuskriptet. Denne forskning og udvikling projektet blev gennemført af forfatterne på David Geffen School of Medicine på UCLA og er gjort mulig ved en kontrakt aftale, der blev tildelt, og som administreres af US Army Medical Research &amp; Materiel Kommando (USAMRMC) og Telemedicin &amp; Advanced Technology Research Center (TATRC), ved Fort Detrick, MD under kontrakt Nummer: W81XWH-10-2-0077. Støtten til disse undersøgelser også kom fra NIH EY04067 og en VA Merit anmeldelse (NB). NCB er en VA Career Research Scientist.

Alle forsøg blev udført i overensstemmelse med retningslinjerne for velfærd forsøgsdyr udstedt af US Public Health Service Politik om Human Care og brugen af ​​forsøgsdyr og udvalg Animal Forskning University of California-Los Angeles (UCLA). Mandlige og kvindelige voksne Sprague-Dawley rotter (Charles River Lab, Wilmington, MA) i alderen 3-5 uger blev anvendt. 1. Animalske og Tissue Forberedelse til whole- mount nethinden og Immunohistokemi protokol <ol><li> Forbered følgende ma...

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I denne artikel har vi beskrevet to forskellige teknikker: 1) Immunohistokemi at vise Fluo-4 lokalisering til gangliecellerne i retinale wholemounts, og 2) Calcium billedbehandling til at analysere calcium dynamik i retina-ganglieceller og deres axoner. Immunhistokemi under anvendelse wholemounts er blevet anvendt til at afsløre lokaliseringen af proteiner i gnaver nethinden 20-23. Der er dog nogle begrænsninger. Kvaliteten af ​​farvningen er stærkt afhængig af antistoffet...

Retina-ganglieceller (rGCS) udtrykker L-, N-, P / Q og T-typen VGCC som bestemt ved farmakologisk blokade af disse bestanddele af helcelle Ca kanalisere nuværende 1,2. VGCC er transmembrane multimere proteiner, der er involveret i transmitterfrigivelse, gentranskription, celle regulering og synaptisk plasticitet 3,4,5. Funktionelle VGCCs består af mindst tre særskilte klasser af underenheder: store, transmembrane α 1 poredannende underenheder, der etablerer kanalens biofysiske og farmakologiske egenskaber, primært ekstracellulære ekstra α 2 δ underenheder og intracellulære β underenheder. De to sidstnævnte formular heteromerkomplekser med forskellige α 1 underenheder og ændre gating kinetik og menneskehandel af kanalerne til plasmamembranen 6. I de seneste årtier har mange teknikker blevet anvendt til at studere protein EXPREssion, såsom immunhistokemi, enzymkoblet immunosorbent-assay, western-analyse og flow-cytometri. Disse teknikker kræver anvendelse af specifikke antistoffer til påvisning af et givet protein af interesse og giver stærke værktøjer til lokalisering og fordeling af specifikke proteiner i forskellige væv. Teknikker, der anvendes til at påvise og kvantificere mRNA-ekspressionsniveauer af et bestemt protein, såsom Northern blot-analyse af RT-PCR, kvantitativ reel-tid RT-PCR, in situ-hybridisering, cDNA microarrays og ribonuklease beskyttelse assay tilvejebringe en alternativ fremgangsmåde, når antistoffer er ikke let til rådighed, eller hvis ekspressionsniveauer af et bestemt protein er lavt 7. Men en begrænsning i anvendelsen af ​​sådanne molekylære teknikker er den krævede identifikation af gensekvensen. For at lokalisere proteiner i nethinden, kan immunhistokemi udføres på whole- mount nethinder. På grund af tilgængeligheden af ​​de rGCS, den whole- mountforberedelse giver en fremragende platform til at studere lokalisering af specifikke proteiner til RGC somata og deres axoner. Ud over deres lokalisering kan visse funktionelle egenskaber VGCCs i rGCS påvises ved anvendelse af calcium billeddannende teknikker. Vi beskriver en calcium imaging protokol til selektivt at mærke rGCS med en calcium indikatorfarvestof til måling af intracellulær calcium dynamik. Bidraget af forskellige VGCCs til calcium signal i forskellige cellulære rum kan isoleres ved anvendelse af undertype-specifikke Ca-kanalblokkere. Måske en af ​​de mest fordelagtige aspekter af calcium imaging her beskrevne teknik, er evnen til samtidigt og uafhængigt optage fra flere rGCS og deres axoner. Selvom mange fysiologiske teknikker, såsom hel celle patch clamp optagelse, giver høj tidslig optagelser Resolution af membranstrømme, det somatiske eller axonal kilde THESe strømme optaget, ikke kan diskrimineres og optagelser kan kun foretages fra en enkelt neuron ad gangen. Multielectrode arrays (MEA) er i stand til samtidigt at optage pigge fra mange celler, men kan hverken opdage eller diskriminere aktivering af for eksempel forskellige undertyper af calciumkanalerne. MEA fortrinsvis optage fra celler, der er placeret i umiddelbar nærhed af en given elektrode 8 og optage fra celler, der frembringer store pigge 9. Optiske billeddiagnostiske metoder giver en alternativ strategi, hvormed de samtidige og uafhængige optagelser af hele populationer af celler, der kan integreres med oplysningerne fra enkelt celle mikroelektrode og patch clamp optagelse og MEA optagelse. Selvom calcium billeddiagnostiske teknikker er beskrevet her var ansat til at studere calcium dynamik rGCS kan patch clamp og multilaterale miljøaftaler også anvendes parallelt med yderligere at belyse de ioniske strømme og standardtilsætningsforsøg egenskaber rGCS. <pclass = &quot;jove_content&quot;&gt; Da fordrevet amacrine celler udgør omkring 60% af den neuronale population i ganglion cellelag i mus nethinden 10, var vores mål at bruge en belastning teknik, der selektivt mærker rGCS med en syntetisk calcium indikatorfarvestof i en whole- mount præparat. Selv syntetiske calcium indikatorfarvestoffer giver en fremragende platform for studiet af intracellulære calcium dynamik, har den udbredte anvendelse blevet hæmmet af den manglende evne til effektivt at indlæse specifikke populationer af neuroner inden for et givet netværk. Mange teknikker såsom isætning 11 og elektroporation 8,12 er blevet udført for at indlæse hele populationer af celler imidlertid sådanne teknikker ikke diskriminerer mellem specifikke celletyper. Genetisk kodet calcium indikatorer giver evnen til selektivt at mærke specifikke populationer af celler imidlertid sådanne metoder kræver frembringelsen af transgene dyr 13. Vor teknik beskriver en method til selektivt at mærke rGCS i whole- mount forberedelse via synsnerven stub injektion af en calcium indikatorfarvestof. Taget sammen, de strukturelle og fysiologiske teknikker beskrevet i denne artikel giver en platform for at studere lokalisering og bidrag VGCCs til calcium signal i rGCS og deres axoner.

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			<td height="21" style="height:21px;width:216px;">Straight scissors</td>
			<td style="width:112px;">WPI</td>
			<td style="width:119px;">14124-G</td>
			<td style="width:229px;">dissecting tools</td>
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			<td height="21" style="height:21px;">Straight Forceps</td>
			<td style="width:112px;">WPI</td>
			<td style="width:119px;">501985</td>
			<td>dissecting tools</td>
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			<td height="21" style="height:21px;width:216px;">curved iris scissors</td>
			<td style="width:112px;">WPI</td>
			<td>504487</td>
			<td>dissecting tools</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cellulose filter paper</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:119px;">HABP04700</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Hibernate A</td>
			<td>Invitrogen</td>
			<td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=A1247501">A12475-01</a></td>
			<td>Media</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vectashield</td>
			<td>Vector</td>
			<td>H-1000&#160;</td>
			<td><a href="https://www.google.com/url?sa=t&amp;rct=j&amp;q=&amp;esrc=s&amp;source=web&amp;cd=1&amp;cad=rja&amp;sqi=2&amp;ved=0CC0QFjAA&amp;url=http%3A%2F%2Fwww.vectorlabs.com%2Fcatalog.aspx%3FcatID%3D279&amp;ei=VAHbUb_fBceeiQLK2IHIDw&amp;usg=AFQjCNHijFTTB8p19FMPQ5hg_qTawXU9mA&amp;sig2=FJCfB9fzKR_TmjxoGvhLcg&amp;bvm=bv.48705608,d.cGE">Mounting Media for Fluorescence</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4 pentapotassium</td>
			<td>Invitrogen</td>
			<td>F-14200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td>Abbott Laboratories</td>
			<td>05260-05</td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope slide</td>
			<td style="width:112px;">Fisher Scientific</td>
			<td style="width:119px;">22-178-277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss LSM 5 Pascal microscope</td>
			<td style="width:112px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axioplan 40x (NA 0.8) objective lens</td>
			<td style="width:112px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:112px;">Molecular Probes</td>
			<td style="width:119px;">A-11034</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RBPMS</td>
			<td style="width:112px;">ProSci, Poway, CA</td>
			<td style="width:119px;"></td>
			<td>A rabbit polyclonal antibody&#160;was generated against the N-terminus of the RBPMS&#160;polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).</td>
		</tr>
	</tbody>
</table>

immunohistochemical and calcium imaging methods in wholemount rat retina

I denne artikel beskriver vi de redskaber, reagenser og de praktiske skridt, der er nødvendige for: 1) vellykket forberedelse af whole- mount nethinder for immunhistokemi og 2) calcium billeddannelse til undersøgelse af spænding gated calcium kanal (VGCC) medieret calcium signalering i retina ganglieceller. Calcium imaging metode beskriver vi omgår spørgsmål vedrørende ikke-specifik belastning af fordrevne amacrine celler i ganglion cellelag.

Immunolabeling med specifikke antistoffer giver en platform for at studere lokaliseringen af ​​bestemte proteiner af interesse i nethinden og calcium imaging teknik tillader studiet af bidraget fra de VGCCs til calcium dynamik i de retinale ganglieceller og deres axoner. Ved anvendelse af et antistof mod ganglioncelle protein RBPMS (RNA-bindende protein med flere splejsning), som selektivt mærker rGCS 19, var vi i stand til at vise, at Fluo-4 mærkningen er begrænset til gan...

Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Immunhistokemi protokoller anvendt til at undersøge lokalisering af et specifikt protein i nethinden. Calcium billeddiagnostiske teknikker er ansat til at studere calcium dynamik i retina-ganglieceller og deres axoner.

Immunhistokemiske og Calcium Imaging Methods in whole- mount rotteretina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Research

JoVE Journal

Neuroscience

Functional Calcium Imaging in Developing Cortical Networks

Funktionel Calcium Imaging i udviklingslandene Kortikal Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging i C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistokemisk analyse i Rat centralnervesystemet og det perifere lymfeknude Vævssnit

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

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Ikke-invasiv Parenkymalt, vaskulære og metaboliske højfrekvent ultralyd og Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Transpupillær to-foton In vivo Billeddannelse af musens nethinde

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging i Mus Ringere Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Hjerne pericyt calcium og hæmodynamisk billeddannelse i transgene mus In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Subcellulær billeddannelse af neuronal calciumhåndtering in vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus (Video) | JoVE

Calciumbilleddannelse i mus overlegen colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging (Video) | JoVE

Calciumbilleddannelse i elektrisk stimulerede fladmonterede nethinden

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...