Name: stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment
Uploaded: 2017-09-01T14:00:00
Description: Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

Forfatterne er Keali Röhm, Louisa Frieß og Heike Menzel taknemmelig for deres ekspert teknisk bistand; til Helga Brünner dyrs pleje; og at Chistopher S. Ward til produktion og distribution af optisk disector gitter plugin for ImageJ software.

alle gældende internationale, nationale og/eller institutionelle retningslinjer for pleje og brug af dyr blev fulgt. Protokollen blev godkendt af lokale myndigheder på Regierung von Unterfranken, Wuerzburg, Tyskland.
 1. behandling af væv og Immunhistokemi <ol><li>aflive mus med CO 2 eller enhver anden godkendt metode.</li>
	<li>Perfuse seks 12-uge-forhenværende C56Bl/6N mand mus transcardially med 10 mL af 0,1 M fosfatbufferet saltopløsning (PBS) ved hjælp af en 25-G kanyl...

<ol>
	<li>Ip, C. W., 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=AAV1/2-induced+overexpression+of+A53T-alpha-synuclein+in+the+substantia+nigra+results+in+degeneration+of+the+nigrostriatal+system+with+Lewy-like+pathology+and+motor+impairment:+a+new+mouse+model+for+Parkinson's+disease.">AAV1/2-induced overexpression of A53T-alpha-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.</a> Acta Neuropathol Commun. 5 (1), 11 (2017).</li><li>Ip, C. W., Beck, S. 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D., Rohrlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+sectioned+cells+via+mathematical+reconstruction.">Counting sectioned cells via mathematical reconstruction.</a> J Comp Neurol. 263 (3), 365-386 (1987).</li><li>Weibel, E. R., Gomez, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+principle+for+counting+tissue+structures+on+random+sections.">A principle for counting tissue structures on random sections.</a> J Appl Physiol. 17, 343-348 (1962).</li><li>West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereological+methods+for+counting+neurons.">Design-based stereological methods for counting neurons.</a> Prog Brain Res. 135, 43-51 (2002).</li><li>Keuker, J. I., Vollmann-Honsdorf, G. K., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+use+the+optical+fractionator:+an+example+based+on+the+estimation+of+neurons+in+the+hippocampal+CA1+and+CA3+regions+of+tree+shrews.">How to use the optical fractionator: an example based on the estimation of neurons in the hippocampal CA1 and CA3 regions of tree shrews.</a> Brain Res Brain Res Protoc. 7 (3), 211-221 (2001).</li><li>Mouton, P. 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Grid_Overlay.java Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/graphic-overlay.html">https://imagej.nih.gov/ij/plugins/graphic-overlay.html</a> (2010)</li><li>. cell_counter.jar Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/cell-counter.html">https://imagej.nih.gov/ij/plugins/cell-counter.html</a> (2010)</li><li>Baquet, Z. C., Williams, D., Brody, J., Smeyne, R. 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+comparison+of+model-based+(2D)+and+design-based+(3D)+stereological+methods+for+estimating+cell+number+in+the+substantia+nigra+pars+compacta+(SNpc)+of+the+C57BL/6J+mouse.">A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse.</a> Neuroscience. 161 (4), 1082-1090 (2009).</li><li>Fu, Y., 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=A+cytoarchitectonic+and+chemoarchitectonic+analysis+of+the+dopamine+cell+groups+in+the+substantia+nigra,+ventral+tegmental+area,+and+retrorubral+field+in+the+mouse.">A cytoarchitectonic and chemoarchitectonic analysis of the dopamine cell groups in the substantia nigra, ventral tegmental area, and retrorubral field in the mouse.</a> Brain Struct Funct. 217 (2), 591-612 (2012).</li><li>German, D. 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Stereologi starter med væv behandling. Den serielle skæring af SN væv skal udføres omhyggeligt for at forhindre tab af dele under stereological analyse. Derudover er et afgørende skridt til at markere en halvkugle for at skelne højre fra venstre SN, når du udfører Stereologi. Placere et lille hul på den øverste del af hjernestammen genereret de bedste resultater i de præsenterede undersøgelse. Desuden siden arbejdet med de optiske fraktioneringskolonne metode kræver, at væv er skåret i tykke sektioner af c...

Kvantificering af neuronal celle nummer er afgørende i prækliniske Parkinsons sygdom forskning for at fastslå omfanget af neurodegeneration substantia nigra (SN)1,2. Den uvildige stereological estimering af celle nummer i en region af interesse betragtes som guldstandarden3,4,5.
Før fremkomsten af uvildige Stereologi, blev antallet af neuroner i sektioner vurderet ved at manipulere optalte celle profiler at korrigere for de variable sandsynligheder at neuroner kommer til syne i en sektion. En af de mest almindeligt anvendte metoder var korrektion af kvantificerede celletal beskrevet af Abercrombie6. Denne metode har forsøgt at tage i betragtning at celler kan kvantificeres mere end én gang, hvis fragmenter af den samme celle findes i tilstødende tynde sektioner. Derfor, Abercrombie og andre forfattere genereres ligninger, der kræves antagelser om form, størrelse og retning af optalte celler7,8. Disse antagelser var dog normalt ikke indså og derfor førte til systematisk fejl og afvigelse fra det faktiske celle nummer (dvs. bias). Desuden kunne bias ikke reduceres ved yderligere prøveudtagning3.
Til stereological vurdering af celle numre ved hjælp af den optiske fraktioneringskolonne der matematiske principper anvendes direkte anslå celle tal direkte i en defineret, 3-dimensionelle volumen. Fordelen ved denne metode er, at det ikke indebærer antagelser om form, størrelse og retning af celler der tælles. Således, den anslåede celle numre er tættere på de sande værdier og komme tættere stikprøvestørrelsen stiger (dvs, neutral)3. Fordi mange regler skal følges, når du bruger Stereologi for at holde metoden uvildig, klar-til-brug kommercielle Stereologi systemer har udviklet (til gennemsyn, se Schmitz og Hof, 20054). Specialiserede Stereologi systemer gennemføre design-baseret stereological metoder med en på forhånd defineret sonder og prøvetagningsordninger stereological vurderinger, der fører til uafhængighed fra form, størrelse, geografiske fordeling og orientering af de celler skal være analyseret4,9. Dog er kommercielt tilgængelige Stereologi systemer dyre; Dette kan begrænse gennemførelsen i ny forskning.
Formålet med denne undersøgelse var at udvikle en brugbar teknik til design-baseret stereological vurdering af dopaminerge celletal i mus SN, beskæftiger metoden optisk fraktioneringskolonne og ved hjælp af standard mikroskopi udstyr (dvs., lys mikroskop, standard mikroskop software og en motoriseret x, y, z fase). Dette præsenteres en trinvis vejledning om hvordan man behandler mus hjernevæv og skøn SN celle numre ved hjælp af design-baseret upartiske Stereologi. Derudover leveres skabeloner for beregningen af den anslåede celle numre og koefficienter af fejl (CE).
Metoden beskrevet her, er ikke begrænset til analysen af SN, men kan tilpasses til brug i andre anatomisk definerede regioner af hjernen, mus eller rotte. Eksempelvis er upartiske Stereologi blevet brugt til at estimere neuronal celle numre i hippocampus10 og locus coeruleus11. Derudover kan celletyper end neuroner, såsom astrocytter12 og mikroglia13, vurderes som godt. Derfor, denne metode kan være nyttig til forskere, der har til hensigt at iværksætte upartiske Stereologi i deres forskning, men er ikke villig til at bruge en masse penge til at købe en Stereologi system.

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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Paxinos mouse atlas</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">The Mouse Brain George Paxinos Keith B.J.FranklinCopyright @2001 by Academic Press CD Rom designet &#38; created by Paul Halasz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">brain matrix slicer mouse</td>
			<td style="width:217px;">Zivic Instruments&#160;</td>
			<td style="width:119px;">BSMAS 001-1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040051000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sucrose /D(+) Saccharose&#160;</td>
			<td style="width:217px;">Roth</td>
			<td style="width:119px;">4621.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isopentane</td>
			<td style="width:217px;">Roth</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glycerol</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040931000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">32205-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:217px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
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			<td height="42" style="height:42px;width:216px;">phosphate buffered saline ingredients:</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">potassium dihydrogen phosphate</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1048731000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">di-sodium hydrogen phosphate dihydrate</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1065801000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">potassium chloride</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1049360500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">normal goat serum</td>
			<td style="width:217px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin</td>
			<td style="width:217px;">Sigma&#160;</td>
			<td style="width:119px;">A4503-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">X100-100ml</td>
			<td>detergent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">3,3-Diaminobenzidine-tetrahydrochlorid/DAB tablets 10mg pH 7.0</td>
			<td style="width:217px;">Kem En Tec</td>
			<td style="width:119px;">4170</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">H2O2/ Hydrogen peroxide 30%</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1072090250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">avidin/biotin reagent</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">32050</td>
			<td>Standard Ultra Sensitive ABC Staining Kit, 1:100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">rabbit anti mouse tyrosine hydroxylase antibody</td>
			<td style="width:217px;">abcam</td>
			<td style="width:119px;">Ab112</td>
			<td>1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">biotinylated goat-anti-rabbit IgG H+L</td>
			<td style="width:217px;">vector laboratories</td>
			<td style="width:119px;">BA-1000</td>
			<td>1:100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">StereoInvestigator version 11.07</td>
			<td style="width:217px;">MBF</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BX53 microscope</td>
			<td style="width:217px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Visiview</td>
			<td style="width:217px;">Visitron Systems GmbH</td>
			<td style="width:119px;">3.3.0.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axiophot2</td>
			<td style="width:217px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ software</td>
			<td style="width:217px;">NIH</td>
			<td style="width:119px;">Version 4.7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-TEK OCT</td>
			<td style="width:217px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dry ice</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">grid overlay plugin</td>
			<td style="width:217px;">Wayne Rasband</td>
			<td style="width:119px;"></td>
			<td>https://imagej.nih.gov/ij/plugins/graphic-overlay.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cell counter plugin</td>
			<td style="width:217px;">Kurt de Vos</td>
			<td style="width:119px;"></td>
			<td>https://imagej.nih.gov/ij/plugins/cell-counter.html).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">optical disector macro</td>
			<td style="width:217px;">Christopher Ward</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57Bl/6N male mice</td>
			<td style="width:217px;">Charles River, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SuperFrost Plus coated object slides</td>
			<td style="width:217px;">Langenbrinck, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needle Microlance 3</td>
			<td style="width:217px;">BD&#160;</td>
			<td align="right" style="width:119px;">300400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">REGLO Analog Infusion pump</td>
			<td style="width:217px;">Ismatec</td>
			<td style="width:119px;">ISM 829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">StereoInvestigator system</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">StereoInvestigator version 11.07</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BX53 microscope</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">BX53 microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">self-assorted stereology</td>
			<td style="width:217px;"></td>
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			<td style="width:467px;">Visiview</td>
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			<td height="21" style="height:21px;width:216px;">Axiophot2</td>
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			<td style="width:119px;"></td>
			<td style="width:467px;">Axiophot2</td>
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stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

I prækliniske Parkinsons sygdom forskning er analyse af nigrostriatal tarmkanalen, herunder kvantificering af dopaminerge neuron tab inden for substantia nigra, afgørende. For at anslå det samlede dopaminerge neuron nummer, anses upartiske Stereologi ved hjælp af metoden optisk fraktioneringskolonne i øjeblikket guldstandarden. Fordi teorien bag metoden optisk fraktioneringskolonne er komplekse og Stereologi er vanskeligt at opnå uden specialudstyr, eksisterer flere kommercielt tilgængelige komplet Stereologi systemer, der indeholder den nødvendige software, rent for celle tælle grundene. Da køber en specialiseret Stereologi opsætning ikke er altid muligt, af mange grunde, denne rapport beskriver en metode for stereological vurdering af dopaminerge neuronal celle tæller ved hjælp af standard mikroskopi udstyr, herunder et lysmikroskop, en motoriseret objekttabellen (x, y, z flyet) med software til billedbehandling og en computer til analyse. En trin for trin forklaring gives på hvordan man udfører stereological kvantificering ved hjælp af metoden optisk fraktioneringskolonne, og præ-programmerede filer til beregning af forventede celletal leveres. For at vurdere rigtigheden af denne metode, blev en sammenligning med data fra et kommercielt tilgængelige Stereologi apparat udført. Sammenlignelige celle numre blev fundet ved hjælp af denne protokol og Stereologi enhed, dermed demonstrere præcisionen af denne protokol for uvildig Stereologi.

Ved hjælp af metoden præsenteres, det anslåede antal af TH + dopaminerge neuroner SN varierede mellem 7,363 og 7,987 celler til højre og venstre SN, mellem 7,446 og 7,904 celler. Det gennemsnitlige antal dopaminerge neuroner (± SEM) var således 7,647 ± 83 celler til højre SN og 7,675 ± 66 til venstre SN. Den beregnede CE for hvert dyr var lavere end 0,08 (range: 0.073-0.079) (figur 7). For at sikre sammenligneligheden af denne metode med kommercielt ...

Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

Dette arbejde præsenterer en trinvis protokol for den uvildige stereological estimering af dopaminerge neuronal celle numre i mus substantia nigra ved hjælp af standard mikroskopi udstyr (dvs. et lysmikroskop en motoriseret objekttabellen (x, y, z fly), og public domain-software til digital billedanalyse.

Stereological vurdering af dopaminerge Neuron antallet i mus Substantia Nigra ved hjælp af optisk fraktioneringskolonne og Standard mikroskopi udstyr

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

The overall goal of this protocol is to describe the design-based stereological estimation of dopaminergic cell number in the mouse SN using standard microscopy equipment. This method is useful to estimate the number of dopaminergic neurons in the mouse substantia nigra for preclinical Parkinson's disease research using design-based unbiased stereology. The main advantage of this technique is that it can be accomplished with standard microscopy equipment without need for a commercially available complete stereology setup.Demonstrating the procedure will be Louisa Friez, a technician from my laboratory. Begin by placing a paraformaldehyde fixed mouse brain into a brain matrix and making a cut plus 0.74 millimeters from bregma. Transfer the caudal part of the brain including the SN into 4%PFA in 0.1 molar and fix overnight at four degrees Celsius.The next day, exchange the PFA for 30%sucrose in 0.1 molar PBS and incubate for another two days at four degrees Celsius to cryo protect the sample. After two days when the brain has sunk in the sucrose, place the tissue into a Cryomold filled with optimal cutting temperature compound and slowly freeze the tissue in liquid dry ice cooled isopentane. Use a cryostat to serially cut 30 micron cryo sections in the coronal plane.Mark the right hemisphere by puncturing a small hole into the upper region of the brain stem. Collect four series of sections starting at minus 2.46 millimeters from bregma and ending at 4.04 millimeters from bregma. Transfer each series of sections into a tube filled with cryoprotectant and store at minus 20 degrees Celsius.Capture digital images of tyrosine hydroxylase immunohistochemically stained SN sections using imaging software coupled to a microscope. Start by determining the actual mount and thickness of the sections at three randomly selected counting sites per section. First click on view top offset to define the top of the section.Then click on stop view top. Thereafter, click on view bottom offset and then click on stop view bottom. Write down the thickness of the section.Calculate the guard zone by subtracting three microns from the top offset and type the result into the top offset box. Then subtract the 13 micron height of the optical dissector from the top offset number and type the result into the bottom offset box. Select the following parameters, steps 14, size 1.00.Select the directory to save the file. Click on sequence to start the acquisition. After the images have been acquired, click on processing and select the following parameters, all planes from sequence, montage and the fast and stitching boxes with an image overlap of 10%Start to stitch the images by clicking on start.This will generate stack images for analysis. To begin, open the image by clicking on file and open. Set the image scale by using the analyze and set scale command.From this step, change the defined size from pixels to microns. Select the plugins then grid then grid type lines command to randomly insert a grid. Check the random offset box.Define the size of one square within the grid as 130 microns by 130 microns to equal 16, 900 square microns. Change image type from 16-bit to RGB color by clicking on image then type then RGB color. Locate the substantia nigra pars compacta.Encircle the region with the paintbrush tool using a brush width of 11. Undo the set scale command by clicking on analyze then set scale then click to remove scale to calculate in pixels rather than microns. Take a screenshot, save the image file and open the new file with ImageJ.Select brush and a brush width of 25. Mark every grid square that contacts the SN for placement of an optical dissector. Select one grid square that includes parts of the SN.Measure the upper left corner of the square with the cursor in ImageJ to define the starting x and y coordinates.Select the spreadsheet template entitled optical dissector position and calculate the optical dissector position. Then estimate the cell number using the calculation of cell count spreadsheet as described in the written portion of the protocol. This low power image shows a section of mouse brain after TH staining for dopaminergic neurons.Higher magnification depicts TH positive dopaminergic neurons. The inset shows the boxed region at increased magnification. This image shows one series of TH stained SN sections covering the whole mouse SN from rostral to caudal.Each section is separated from the consecutive section by 120 microns. The number of TH positive neurons in the right and left mouse SN were estimated using the method shown in this video and using a commercially available stereology system. As seen here, both methods resulted in comparable cell counts and there were no significant differences between data sets.Don't forget that working with paraformaldehyde can be extremely hazardous and precautions such as wearing of personal protection equipment like protective gloves should always be taken while performing this procedure. After watching this video, you should have a good understanding of how to perform unbiased stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fraction method and standard microscopy equipment.

Research

JoVE Journal

Neuroscience

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