Name: detection of abnormal prion protein by immunohistochemistry
Uploaded: 2023-05-05T14:20:00
Description: Watch this Scientific Journal Video about Detection of Abnormal Prion Protein by Immunohistochemistry at JoVE.com

Denna artikel finansierades av projektet POCI-01-0145-FEDER-029947 "Chronic wasting disease risk assessment in Portugal" med stöd av FCT (Fundação para a Ciência e a Tecnologia) - FEDER -Balcão2020. Författarna till forskningsenheten CECAV fick också finansiering från FCT, under projektet UIDB/CVT/0772/2020.Vi tackar Bruce C. Campbell, forskningsdirektör (pensionerad), Western Regional Research Center, USDA, för hans hjälp.

IHC-förfarandet som beskrivs här är en del av forskningsprojektet FAIRJ-CT98-7021, "Inrättandet av ett europeiskt nätverk för övervakning av TSE hos idisslare och standardisering och harmonisering av processen och kriterierna för identifiering av misstänkta fall". Den följer de diagnostiska kriterier som fastställts av Världsorganisationen för djurhälsa, WOAH (tidigare Office International des Épizooties, OIE)1 och European Reference Laboratory for animal TSE...

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	<li>. WOAH, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Online Access. Chapter 3.4.5.-Bovine Spongiform Encephalopathy (Version May 2021) and Chapter 3.8.11. - Scrapie (Version May 2022) Available from: <a target="_blank" href="https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-manual-online-access/">https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-manual-online-access/</a> (2022)</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+methods+of+immunohistochemistry.">Principles and methods of immunohistochemistry.</a> Methods in Molecular Biology. 1641, 115-128 (2017).</li><li>Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M., Oliver, C., Jamur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-Induced+antigen+retrieval+for+immunohistochemical+reactions+in+routinely+processed+paraffin+sections.+Immunocytochemical+Methods+and+Protocols.">Heat-Induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections. Immunocytochemical Methods and Protocols.</a> Methods in Molecular Biology. 588, 103-119 (2010).</li><li>Duraiyan, J., Govindarajan, R., Kaliyappan, K., Palanisamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+immunohistochemistry).">Applications of immunohistochemistry).</a> Journal Pharmacy Bioallied Sciences. 4, 307-309 (2012).</li><li>Orge, 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=Neuropathology+of+Animal+Prion+Diseases.">Neuropathology of Animal Prion Diseases.</a> Biomolecules. 11 (3), 466 (2021).</li><li>. Biosafety in Microbiological and Biomedical Laboratories. 6th Edition Revised June 2020 Available from: <a target="_blank" href="https://www.cdc.gov/labs/pdf/SF_19_308133-A_BMBL6_00-BOOK-WEB-final-3.pdf">https://www.cdc.gov/labs/pdf/SF_19_308133-A_BMBL6_00-BOOK-WEB-final-3.pdf</a> (2022)</li><li>APHA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation,+tissue+processing,+histology+and+immunohistochemistry+procedures+for+diagnosis+of+animal+TSE+(BSE,+scrapie,+atypical+scrapie,+CWD).+Histo+&amp;+IHC+protocols+for+TSE+diagnosis_Rev_Jan2019.pdf.">Fixation, tissue processing, histology and immunohistochemistry procedures for diagnosis of animal TSE (BSE, scrapie, atypical scrapie, CWD). Histo &amp; IHC protocols for TSE diagnosis_Rev_Jan2019.pdf.</a> TSEglobalNet - International Reference Laboratory for TSE. , (2022).</li><li>Sample requirements for TSE testing and confirmation. Version 1.0. TSE EURL Available from: <a target="_blank" href="https://www.izsplv.it/it/istituto/212-centri-eccellenza/laboratori-internazionali-riferimento/422-eurl_tses.html">https://www.izsplv.it/it/istituto/212-centri-eccellenza/laboratori-internazionali-riferimento/422-eurl_tses.html</a> (2019)</li><li>TSE EU Reference Laboratory Guidelines for the detection of Chronic Wasting Disease in cervids. Version 1.0. TSE EURL Available from: <a target="_blank" href="https://www.izsplv.it/it/istituto/212-centri-eccellenza/laboratori-internazionali-riferimento/422-eurl_tses.html">https://www.izsplv.it/it/istituto/212-centri-eccellenza/laboratori-internazionali-riferimento/422-eurl_tses.html</a> (2019)</li><li>Machado, C. N., 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=TSE+Monitoring+in+Wildlife+Epidemiology,+Transmission,+Diagnosis,+Genetics+and+Control.">TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control.</a> Wildlife Population Monitoring. IntechOpen. , (2019).</li><li>APHA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathology:+Confirmatory+diagnosis+of+transmissible+spongiform+encephalopathies+(TSEs)+in+cattle+and+small+ruminants.+Confirmatory+(Histo+&amp;+IHC)+diagnostic+criteria+Rev_Jan2019.pdf.">Neuropathology: Confirmatory diagnosis of transmissible spongiform encephalopathies (TSEs) in cattle and small ruminants. Confirmatory (Histo &amp; IHC) diagnostic criteria Rev_Jan2019.pdf.</a> TSEglobalNet - International Reference Laboratory for TSE. , (2019).</li><li>Ryder, S. J., Spencer, Y. I., Bellerby, P. J., March, S. A. <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+PrP+in+the+medulla+oblongata+of+sheep:+The+spectrum+of+staining+in+normal+and+scrapie-affected+sheep.">Immunohistochemical detection of PrP in the medulla oblongata of sheep: The spectrum of staining in normal and scrapie-affected sheep.</a> The Veterinary Record. 148 (1), 7-13 (2001).</li><li>Simmons, 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=Experimental+classical+bovine+spongiform+encephalopathy:+definition+and+progression+of+neural+PrP+immunolabeling+in+relation+to+diagnosis+and+disease+controls.">Experimental classical bovine spongiform encephalopathy: definition and progression of neural PrP immunolabeling in relation to diagnosis and disease controls.</a> Veterinary Pathology. 48 (5), 948-963 (2011).</li><li>Orge, 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=Identification+of+H-type+BSE+in+Portugal.">Identification of H-type BSE in Portugal.</a> Prion. 9 (1), 22-28 (2015).</li><li>Orge, L., Simas, J. P., Fernandes, A. C., Ramos, M., Galo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarity+of+the+lesion+profile+of+BSE+in+Portuguese+cattle+to+that+described+in+British+cattle.">Similarity of the lesion profile of BSE in Portuguese cattle to that described in British cattle.</a> Veterinary Record. 147 (17), 486-488 (2000).</li><li>Pires, M. A., Travassos, F. S., G&#228;rtner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atlas+of+veterinary+pathology.">Atlas of veterinary pathology.</a> Biopathology. Lidel VII. 195 (6), 179-180 (2004).</li><li>Pires, M. 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=Immunology+protocols,+didactic+series.">Immunology protocols, didactic series.</a> Applied Sciences. , 357 (2010).</li></ol>

TSE är potentiella zoonotiska sjukdomar. Efter uppkomsten av BSE 1986 i Förenade kungariket blev Portugal en av Europeiska unionens medlemsstater med en högre förekomst av denna sjukdom14,15. För att bekämpa denna sjukdom, andra TSE som har uppstått (klassisk och atypisk skrapie, BSE-varianter och för närvarande övervakning av chronic wasting disease hos hjortdjur), har övervakningsmekanismer utvecklats av generaldirektoratet för livsmedel och veterin...

Enligt prionhypotesen är den onormala isoformen (PrPSc) den primära eller enda komponenten i smittämnet vid transmissibel spongiform encefalopati (TSE). Bekräftelse för diagnos av TSE bygger på immunodetektion av PrPSc genom tillämpning av immunhistokemiprotokoll (IHC) och/eller västerländska immunoblotmetoder (WB) av encefalonvävnader1.
IHC är en metod som använder monoklonala eller i vissa fall polyklonala antikroppar (som primära antikroppar) som ett första steg i immunfärgning av specifikt riktade antigener av intresse belägna i celler i en vävnadssektion. Eventuell effektiv primär antikropps-antigenbindning visualiseras sedan med hjälp av sekundära antikroppar specifika för de primära antikropparna. Dessa sekundära antikroppar är konjugerade till enzymer, såsom pepparrotsperoxidas (HRP) eller alkaliskt fosfatas (AP). Visualisering uppnås sedan genom att tillsätta ett substrat till dessa enzymer, vilket ger olösliga färgprodukter lokaliserade i områden där de primära antikropparna är bundna till de riktade antigenerna. Förbättrad visualisering kan uppnås genom motfärgning, där ett färgämne används för att skapa en kontrast mellan immunomärkt och icke-immunmärkt vävnad2.
Med IHC med användning av formalinfixerade paraffininbäddade vävnader (FFPE) kan formalinfixering upphäva effektiviteten hos primära antikroppar på grund av tvärbindning genom formaldehyd och uppvärmning och uttorkning under paraffininbäddning. Dessa förändrar konformationen av proteiner, förstör, denaturerar eller maskerar epitoperna, vilket minskar eller upphäver deras upptäckt3. Som sådan kräver detta antigenhämtning (AR). AR-teknikerna stör formaldehydrelaterad kemisk grupptvärbindning i de antigena molekylerna, vilket återställer eller demaskerar den ursprungliga antigen-proteinkonformationen. Detta resulterar i återvinning av antikropps-antigen (epitop) affinitet för immunmärkning. Den slutliga effekten av AR beror på egenskaperna hos det riktade antigenet och/eller den primära antikroppen2.
Värmeinducerad antigen (epitop) hämtning (HIER) är en procedur av AR3 och används rutinmässigt för PrPSc IHC-detektion, som beskrivs häri. IHC är viktigt för diagnos och används i forskningslaboratorier för att bestämma vävnadsdistributionen av ett patologiassocierat antigen. Det används ofta för att diagnostisera och undersöka cancer, neurovetenskap och infektionssjukdomar4, bland andra. För TSE spelar IHC en viktig roll i diagnos och forskning för att bekräfta och undersöka PrPSc-distribution i naturliga värdar och experimentella modeller. IHC bidrar till prionpatogenesstudier och analys av PrP Sc-deponeringstyper och -mönster, nämligen i neurala vävnader5, för att upptäcka avvikelser från rutinmässigt beskrivna infektioner och identifiera förmodade nya prionstammar.
Eftersom prioner av bovin spongiform encefalopati (BSE) kan infektera människor, kan vissa laboratorieprotokoll som ingår i arbetet med BSE kräva användning av BSL-3-anläggningar och -metoder6. Dessa inkluderar användning av en förseglad sekundär behållare för att transportera potentiella BSE-infekterade vävnadsprover inom institutet och laboratoriet. Det omfattar också att utse inneslutningsområden och särskild prionutrustning för forskning och analys av BSE när så är möjligt. Detta görs för att förhindra kontaminering utanför arbetsområdet och ge ett begränsat utrymme eftersom dekontamineringsförfaranden blir nödvändiga.
Följaktligen följer Laboratory of Pathology of INIAV rekommenderade biosäkerhetsnivå-3 (BSL-3) anläggningar och praxis6 för att hantera potentiella prioninfekterade prover av vävnader från nötkreatur, små idisslare och hjortdjur som är associerade med TSE-övervakningen.
Formalinfixerade och paraffininbäddade vävnader som ingår i TSE-diagnostik eller TSE-forskningsförfaranden, särskilt i centrala nervsystemet, kan vara potentiellt smittsamma. Dessa fasta vävnader måste därför behandlas med myrsyra för att minska infektiviteten hos prioner, om sådana förekommer, före vävnadsbearbetningen. Detta utförs genom att placera fasta, trimmade vävnader (ca 2-4 mm tjocklek) i en bearbetningskassett. Kassetten nedsänks sedan i 98% myrsyra (i 1 h). Efter nedsänkning tvättas kassetterna med vävnader i rinnande kranvatten i 30 minuter och återförs till fixeringsmedlet före vidare bearbetning. Om vävnadssnitt inte behandlas före bearbetning måste postmikrotomiska vävnadssnitt nedsänkas i outspädd myrsyra i minst 5 minuter före histologisk färgning7. IHC-protokollet för PrPSc innehåller ett rutinmässigt myrsyraepitop-demasking steg, som också tjänar till att inaktivera prioner7. Efter dessa prioninaktiveringssteg kan de resulterande fasta vävnaderna bearbetas vid BSL-2 med användning av standard BSL-2-praxis.
Det minsta kravet på vävnadsprovtagning för TSE-diagnos hos alla djur som omfattas av TSE-övervakningen är insamling av hjärnstammen (på obexnivå). För att upptäcka atypisk BSE och skrapie rekommenderas dessutom att en del av lillhjärnan också samlas in 1,8. För CWD-diagnos bör både hjärnstammen (obex) och retrofaryngeala lymfkörtlar testas eftersom PrP Sc kan detekteras i lymfoida vävnader utan detekterbar PrPSc i obex9, granskad av Machado et al.10.
Obex-delen av hjärnstammen inkluderar diagnostiska TSE-målplatser, nämligen dorsal vaguskärna (DVN), enkroppskärna (STN) och ryggradskärnan i trigeminusnerven (V). Dessa områden uppvisar konsekvent bilateral ackumulering av PrPSc , även i de tidiga stadierna av BSE och klassisk skrapie. I kliniska fall av avancerad TSE uppvisar alla gråämnesområden inom hjärnstammen utbredd PrPSc-fördelning 11.
Före snittning och bearbetning utvärderas hjärnproverna (figur 1) för att fastställa graden av autolys och förekomsten av eventuell vävnadsskada som potentiellt äventyrar provets lämplighet för IHC-baserad bekräftande diagnos8. För att validera integriteten hos de preparativa protokollen och analysresultaten ingår TSE-positiva och negativa vävnadsprover som kontroller i samband med beredning av vävnader från testfall i varje analys.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg"> Figur 1: PrPSc IHC-förfarandet. Representation som visar steg-för-steg-sekvensen av PrPSc IHC-proceduren från deparaffinisering av vävnadssnitt till eventuell immunfärgning och detektion (FFPE - Formalinfixerad paraffininbäddad; Mab - monoklonal antikropp; DAB - 3,3' diaminobensidin). Denna siffra skapades BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">Klicka här för att se en större version av denna figur.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absolute ethanol&#160;</td>
			<td>Labchem</td>
			<td>LB0507-9010</td>
			<td>Undituled</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Diluted 90%, 70% and 50% in distilled water</td>
		</tr>
		<tr>
			<td>Avidin-biotin complex and peroxidase 
			Vectastain Elite ABC kit Peroxidase</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>Prepare and gently mix 30 min before use according to kit instructions. Do not mix after standing.</td>
		</tr>
		<tr>
			<td>Biotinylated secondary antibody (Horse anti-mouse IgG H+L)&#160;</td>
			<td>Vector Laboratories</td>
			<td>BA-2000-1.5</td>
			<td>Dilute at 1/200 in TBS with 10% horse normal serum. Prepare the volume required depending on the number of sections.</td>
		</tr>
		<tr>
			<td>Chromogen Diaminobenzidine- DAB, substrate kit, Peroxidase&#160;</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>Prepare before use according to kit instructions. 
			Use 400 &#181;L of solution per section.</td>
		</tr>
		<tr>
			<td>DakoCytomation Pascal pressure chamber</td>
			<td>DAKO</td>
			<td>S2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ehrlich&#8217;s Hematoxylin:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Merck</td>
			<td>115938</td>
			<td>Dissolve 2 g of hematoxylin in 100 mL of absolute ethanol. Add 100 mL of distilled water, 10 mL of glacial acetic acid and 15 g of potassium alum with constant stirring. Add 100 mL of glycerin. The natural oxidation process takes 2 months, before use.</td>
		</tr>
		<tr>
			<td>Absolute ethanol&#160;</td>
			<td>Labchem</td>
			<td>LB0507-9010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid&#160;</td>
			<td>Merck</td>
			<td>101830</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium alum</td>
			<td>Merck</td>
			<td>1.01047.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>Merck</td>
			<td>1.04091.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Endogenous Peroxidase Block solution (3% concentration H2O2):</td>
			<td></td>
			<td></td>
			<td>40 mL Hydrogen peroxide (30% w/w) in 360 mL Methanol. 
			Prepare before use</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (30% w/w)</td>
			<td>Scharlau</td>
			<td>HI0136</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>322415-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid 98%</td>
			<td>Merck</td>
			<td>1.00264.1000</td>
			<td>Undiluted</td>
		</tr>
		<tr>
			<td>Microtome&#160;</td>
			<td>Shandon-AS325</td>
			<td>Microtome&#160;</td>
			<td>Shandon-AS325</td>
		</tr>
		<tr>
			<td>Mounting medium Entellan</td>
			<td>Merck</td>
			<td>107960</td>
			<td>Ready- to- use.</td>
		</tr>
		<tr>
			<td>Normal serum (20% ) block solution in TBS: 
			Horse normal serum</td>
			<td> 
			Gibco</td>
			<td> 
			16050-122&#160;</td>
			<td>Prepare final volume according to the number of sections in the assay 
			(200 &#181;L of solution per section).</td>
		</tr>
		<tr>
			<td>Primary antibody anti-PrP Mouse MAb 2G11&#160;</td>
			<td>BIORAD</td>
			<td>MCA2460</td>
			<td>PrP 146-R154R171182 
			Ovine including atypical scrapie, cervine, feline. Not suitable for bovine. 
			According to the number of sections in the assay (200 &#181;L of solution per section) and antibody dilution, prepare final volume in TBS supplemented with 10% of normal serum from the species the secondary antibody was raised in (horse normal serum) 
			Usual antibody dilution: MAb 2G11 1/100 but working dilution should be established in every new batch to get the concentration to give the strongest labelling with lowest background. For storage, freeze aliquot volumes of a minimum of 10 &#956;L into sterile microtubes. Defrost and use one aliquot at a time.&#160;</td>
		</tr>
		<tr>
			<td>Primary antibody anti-PrP Mouse MAb 12F10&#160;</td>
			<td>Cayman&#160; Chemical Company&#160;</td>
			<td>189710</td>
			<td>PrP142-160 
			Bovine, not suitable for ovine 
			Usual antibody dilution: 1/200 but working dilution should also be established. Prepare as MAb 2G11</td>
		</tr>
		<tr>
			<td>Shandon CoverplateTM chamber</td>
			<td>Thermo Scientific&#160;</td>
			<td>72110017</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Sequenza&#174; Immunstaining center&#160;</td>
			<td>Thermo Scientific</td>
			<td>73300001</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Sequenza&#174; Immunstaining slide rack</td>
			<td>Thermo Scientific</td>
			<td>73310017</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution Citrate Buffer (10 mM pH 6.1):</td>
			<td></td>
			<td></td>
			<td>2.55 g Tri-sodium citrate dihydrate&#160; and 0.255 g Citric acid in one litre purified water. 
			Adjust pH of working solution to 6.1 using 10 mM citric acid solution (1.05 g citric acid in 500 mL purified water) 
			Prepare on assay day.&#160;</td>
		</tr>
		<tr>
			<td>Tri-sodium citrate dihydrate</td>
			<td>Sigma-aldrich&#160;</td>
			<td>S4641-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma Aldrich</td>
			<td>C0759</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining jar and basket</td>
			<td>Deltalab&#160;</td>
			<td>19360</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>19361</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus microscope slides</td>
			<td>VWR</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Buffered Saline solution (TBS) (50 mM TRIZMA BASE; 0.8% NaCI; pH 7.6):</td>
			<td></td>
			<td></td>
			<td>10xTBS (stock solution 0.5 M TRIZMA BASE; 8% NaCI; pH 7.6): 
			TRIZMA BASE 60,57 g and NaCl 80 g in 800 mL purified water. Adjust pH of stock solution using Hydrochloric acid 37% and final volume to one litre with purified water (keep 5&#177; 3 &#176;C until 2 months) 
			Dilute TBS stock solution 1/10 on assay day.&#160;</td>
		</tr>
		<tr>
			<td>TRIZMA BASE</td>
			<td>Sigma Aldrich</td>
			<td>T6066-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Merck</td>
			<td>106404</td>
			<td></td>
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		<tr>
			<td>Xylene&#160;</td>
			<td>Panreac Applied Chem ITW reagents</td>
			<td>251769</td>
			<td>Undiluted</td>
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detection of abnormal prion protein by immunohistochemistry

Onormala prionproteiner (PrPSc) är den sjukdomsassocierade isoformen av cellulärt prionprotein och diagnostiska markörer för transmissibel spongiform encefalopati (TSE). Dessa neurodegenerativa sjukdomar drabbar människor och flera djurarter och inkluderar skrapie, zoonotisk bovin spongiform encefalopati (BSE), chronic wasting disease of cervids (CWD) och den nyligen identifierade kamelprionsjukdomen (CPD). Diagnos av TSE bygger på immundetektion av PrPSc genom tillämpning av både immunhistokemi (IHC) och västerländska immunoblotmetoder (WB) på encefalonvävnader, nämligen hjärnstammen (obexnivå). IHC är en allmänt använd metod som använder primära antikroppar (monoklonala eller polyklonala) mot antigener av intresse i celler i en vävnadssektion. Antikropps-antigenbindningen kan visualiseras genom en färgreaktion som förblir lokaliserad i det område av vävnaden eller cellen där antikroppen riktades. Som sådan, i prionsjukdomar, liksom i andra forskningsområden, används immunhistokemiska tekniker inte enbart för diagnostiska ändamål utan också i patogenesstudier. Sådana studier innefattar detektering av PrPSc-mönster och typer från de tidigare beskrivna för att identifiera de nya prionstammarna. Eftersom BSE kan smitta människor rekommenderas att biosäkerhetslaboratorier på nivå 3 (BSL-3) och/eller metoder används för att hantera nötkreatur, mindre idisslare och hjortprover som omfattas av TSE-övervakningen. Dessutom rekommenderas inneslutnings- och prionsärskild utrustning, när så är möjligt, för att begränsa kontaminering. PrPSc IHC-förfarandet består av ett epitoptest av myrsyraepitoper som också fungerar som en prioninaktiveringsåtgärd, eftersom formalinfixerade och paraffininbäddade vävnader som används i denna teknik förblir smittsamma. Vid tolkning av resultaten måste man vara noga med att skilja icke-specifik immunmärkning från målmärkning. För detta ändamål är det viktigt att känna igen artefakter av immunmärkning som erhållits i kända TSE-negativa kontrolldjur för att skilja dem från specifika PrP Sc-immunmärkningstyper, som kan variera mellan TSE-stammar, värdarter och prnp-genotyp, som beskrivs närmare häri.

Med tanke på att icke-specifik målimmunmärkning är möjlig är det viktigt att fastställa nivån av icke-specifik immunmärkning hos kända TSE-negativa kontrolldjur. Detta är ett viktigt steg för att korrekt tolka specifik PrPSc-immunmärkning 7 (figur 2). Icke-målmärkning av anti-PrP-antikroppar har noterats förekomma som diskret intraneuronal finpartikelfärgning (tydligare i hypoglossalkärnan och accessorisk cuneatkärna), oregelbun...

Watch this Scientific Journal Video about Detection of Abnormal Prion Protein by Immunohistochemistry at JoVE.com

Detection of Abnormal Prion Protein by Immunohistochemistry

Immunolabeling onormalt prionprotein med hjälp av immunhistokemiska protokoll kräver specifika prov- och anti-PrP-antikroppsberedningsmetoder. Detta protokoll beskriver de viktigaste stegen i epitopdemaskering för att säkerställa korrekt PrP-immunmärkning och för att minimera icke-specifik bakgrundsfärgning. Detta tillvägagångssätt beaktar också biosäkerhetsåtgärder vid genomförande av immunhistokemiska studier med prioninfekterade vävnader.

Detektion av onormalt prionprotein genom immunhistokemi

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform ...

Tekniken möjliggör bekräftande diagnos av prionsjukdomar med tekniken PrP-skrapie i vävnader, analys av PrP-skrapietyper och distribution för att utvärdera straffavvikelse till den kända PrP-skrapiedeponeringsprofilen. Tekniken är standardiserad och använder specifika antikroppar mot PrP-skrapie, vilket möjliggör visualisering av PrP-skrapieavsättningen i vävnader. Tekniken ger också information om det neuroanatomiska området och typen av celler.Börja med att placera objektglasen med vävnadssektionerna i en rostfri färgningskorg. Fäll ut korgen i ett xylenbad. Efter tre minuter, ta bort och sänk ner korgen igen.För att avlägsna xylen och rehydrera sektionerna, sänk ner korgen i ett absolut etanolbad. Efter tre minuter, ta bort och sänk ner korgen igen. Lufttorka sektionerna innan du lägger dem i ett 90% etanolbad i en minut.Överför sedan sektionen till ett 70% etanolbad i ytterligare en minut. För varje behandling med etanolbad, skaka försiktigt glidkorgen två gånger och töm korgen före nästa överföring. Överför korgen till ett 50% etanolbad i en minut.Sänk försiktigt ner vävnadssektionerna i 98% myrsyra vid rumstemperatur i 30 minuter. Skölj sektionerna i kranvatten i fem minuter följt av sköljning två gånger i destillerat vatten. Använd en färgningsbehållare av rostfritt stål i tryckkammaren fylld med 500 ml destillerat vatten för att förbehandla 10 millimolar citratbuffert vid 98 grader Celsius i 20 minuter.För kvalitetskontrolländamål, placera en sektion av självhäftande autoklavindikatortejp på korgen för att övervaka temperatur och tryck. När larmet indikerar att utrustningen har uppnått programtid och temperatur, sänk ner korgen med bilder. Starta sedan programmet på tryckkammaren till Set Point 2 och registrera initialt och slutligt tryck för detta program.För inaktivering av endogent peroxidas, sänk ner glaskorgen med provsektionerna i ett bad med 3% väteperoxid i metanol i 30 minuter. Överbrygga sektionerna under rinnande vatten i fem minuter Efter tömning, sänk ner sektionerna i Tris-buffrad saltlösning i ytterligare fem minuter. För immundetektion, placera varje glas på en kommersiellt tillgänglig täckplatthållare förfuktad med Tris-buffrad saltlösning med vävnadssidan vänd mot hållaren och glidkanterna sammanfaller med hållarens två nedre punkter.Se till att undvika luftbubblor. Håll glidhållarenheten mellan tummen och pekfingret. Håll ett finger ovanpå provglaset och det andra på undersidan av hållaren och placera sedan enheten i systemets galleri.För att säkerställa att uppsättningen är väl monterad, fyll brunnen mellan provglaset och hållaren med Tris-buffrad saltlösning utan att överflöda den. Från denna punkt, se till att cirka 80 mikroliter Tris-buffrad saltlösning måste hållas kvar mellan hållaren och glaset. Låt inte sektionerna torka.Minska bakgrundsfärgningen före behandling med den primära antikroppen genom att förinkubera objektglasproverna i 30 minuter med 20% normalt serum från samma art som den sekundära antikroppsvärden i Tris-buffrad saltlösning. Utan att tvätta sektionerna, applicera 200 mikroliter av den primära antikroppslösningen direkt i varje brunn i glidhållarsatsen och inkubera i 60 minuter vid rumstemperatur. För att tvätta sektionerna, fyll brunnarna med Tris-buffrad saltlösning och vänta i fem minuter.Upprepa tvätten två gånger. Späd sedan den biotinylerade sekundära antikroppen i koncentrationen 1 till 200 i Tris-buffrad saltlösning med 10% hästserum. Reparera önskad volym beroende på antalet sektioner som ska behandlas.Applicera 200 mikroliter sekundär antikroppslösning på varje brunn i glidhållarsatsen. Efter 30 minuters inkubation vid rumstemperatur, upprepa den Tris-buffrade saltlösningen. För inkubation med avidin-biotinkomplexperoxidas, bereda reagenset 30 minuter före användning och applicera 200 mikroliter av lösningen i varje brunn på glidplatthållaren.När du är klar, inkubera platthållaren i 30 minuter vid rumstemperatur. Nivån av icke-specifik immunmärkning hos kända transmissibla spongiforma encefalopatier-negativa kontrolldjur bestämdes för att tolka de specifika onormala prionproteinernas immunmärkning korrekt. Icke-målmärkning av anti-PrP-antikroppar noterades förekomma som diskret intraneuronal finpartikelfärgning, oregelbundna fina linjära trådar av fläck i neuropilen, diffus icke-partikelmärkning i vissa grå och vita områden och diffus cytoplasmatisk märkning av neuroner.Tabellen sammanfattar beskrivningen av immunmärkta onormala prionproteintyper som observerats vid bovin spongiform encefalopati, klassisk och atypisk skrapie och en chronic wasting disease hos hjortdjur för en positiv diagnos och fastställda kriterier för negativa, ofullständiga och olämpliga resultat. Alla steg är mycket viktiga. Lösningarnas pH och rumstemperaturen är viktiga funktioner för att denna teknik ska lyckas.Att utveckla immunhistokemi för att detektera både PrP-skrapie och typ av celler kan ge information om varje cell som är involverad i primärpatogenes.

detection-of-abnormal-prion-protein-by-immunohistochemistry

Research

JoVE Journal

Neuroscience

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Kombinera lipofila färgämne, På plats Hybridisering, immunohistokemi, och histologi

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Detektion av Microregional hypoxi i mus Cerebral Cortex med två-foton Imaging av endogen NADH fluorescens

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Wholemount Immunohistokemi för att avslöja Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Dissektion och Immunohistokemi av larver, pupal och vuxna<em&gt; Drosophila</em&gt; Näthinnor

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Detektering av protein palmitoylering i odlade hippocampus neuroner genom immunprecipitation och Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Detektion av<em&gt; In situ</em&gt; Protein-proteinkomplex på<em&gt; Drosophila</em&gt; Larv Neuromuskulära förbindelsen Använda Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

Bioluminescence avbildning av Neuroinflammation i transgena möss efter perifer ympning av alfa-synuklein fibriller

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Övervakning Cell till cell överföring av Prion-liknande Protein aggregat i Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Kvantifiera mikroglia morfologi från mikrofotografier av immunhistokemi beredd vävnad med hjälp av ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Kvantitativ autoradiografisk metod för bestämning av regionala frekvenser av cerebral protein syntes in vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...