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

Denne artikkelen ble finansiert av prosjektet POCI-01-0145-FEDER-029947 "Risikovurdering av kronisk sløsende sykdom i Portugal" støttet av FCT (Fundação para a Ciência e a Tecnologia) - FEDER -Balcão2020. Også forfatterne av forskningsenheten CECAV mottok finansiering fra FCT, under prosjektet UIDB / CVT / 0772/2020.Vi takker Bruce C. Campbell, forskningsdirektør (pensjonert), Western Regional Research Center, USDA, for hans hjelp.

IHC-prosedyren beskrevet her er en del av forskningsprosjektet FAIRJ-CT98-7021, "Etablering av et europeisk nettverk for overvåking av drøvtyggende TSE og standardisering og harmonisering av prosessen og kriterier for identifisering av mistenkelige tilfeller". Det følger de diagnostiske kriteriene definert av Verdens dyrehelseorganisasjon, WOAH (tidligere kalt Office International des Épizooties, OIE)1 og European Reference Laboratory for animal TSEs 8,11.<su...

<ol>
	<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 er potensielle zoonotiske sykdommer. Etter fremveksten av BSE i 1986 i Storbritannia, ble Portugal en av EUs medlemsstater med en høyere forekomst av denne sykdommen14,15. For å kontrollere denne sykdommen, andre TSE som har dukket opp (klassisk og atypisk skrapesyke, BSE-varianter, og for tiden overvåking av skrantesyke hos hjortedyr), ble overvåkingsmekanismer utviklet av Generaldirektoratet for mat og veterinær (DGAV) og Nasjonalt institutt for landbr...

Ifølge prionhypotesen er den unormale isoformen (PrPSc) den primære eller eneste komponenten av det smittsomme stoffet i overførbare spongiforme encefalopatier (TSE). Bekreftelse for diagnostisering av TSE er avhengig av immundeteksjon av PrPSc ved bruk av immunhistokjemi (IHC) protokoller og / eller vestlige immunoblotmetoder (WB) av encephalon vev1.
IHC er en metode som benytter monoklonale eller, i noen tilfeller, polyklonale antistoffer (som primære antistoffer) som et første skritt i immunfarging av spesifikt målrettede antigener av interesse lokalisert i celler i en vevsseksjon. Enhver effektiv primær antistoff-antigenbinding visualiseres deretter ved bruk av sekundære antistoffer som er spesifikke for de primære antistoffene. Disse sekundære antistoffene konjugeres til enzymer, slik som pepperrotperoksidase (HRP) eller alkalisk fosfatase (AP). Visualisering oppnås deretter ved å legge til et substrat til disse enzymene, og produsere uoppløselige fargeprodukter lokalisert i områder der de primære antistoffene er bundet til de målrettede antigenene. Forbedret visualisering kan oppnås ved motfarging, hvor et fargestoff brukes til å skape en kontrast mellom immunmerket og ikke-immunmerket vev2.
Med IHC ved bruk av formalinfast parafininnebygd vev (FFPE), kan formalinfiksering oppheve effektiviteten av primære antistoffer på grunn av kryssbinding ved formaldehyd og oppvarming og dehydrering under parafininnbygging. Disse endrer konformasjonen av proteiner, ødelegger, denaturerer eller maskerer epitopene, og reduserer eller opphever dermed deres deteksjon3. Som sådan krever dette antigengjenfinning (AR). AR-teknikkene forstyrrer formaldehydrelatert kjemisk gruppe-kryssbinding i de antigene molekylene, og gjenoppretter eller avdekker dermed den opprinnelige antigen-proteinkonformasjonen. Dette resulterer i gjenoppretting av antistoff-antigen (epitop) affinitet for immunmerking. Den endelige effekten av AR avhenger av egenskapene til det målrettede antigenet og / eller det primære antistoffet2.
Heat-induced antigen (epitope) retrieval (HIER) er en prosedyre for AR3 og brukes rutinemessig for PrPSc IHC-deteksjon, som beskrevet her. IHC er avgjørende for diagnose og brukes i forskningslaboratorier for å bestemme vevsfordelingen av et patologiassosiert antigen. Det er mye brukt i diagnostisering og forskning på kreft, nevrovitenskap og smittsomme sykdommer4, blant andre. For TSE spiller IHC en viktig rolle i diagnostisering og forskning for å bekrefte og undersøke PrPSc-distribusjon i naturlige verter og eksperimentelle modeller. IHC bidrar til prionpatogenesestudier og analyse av PrP Sc-avsetningstyper og mønstre, nemlig i nevrale vev5, for å oppdage avvik fra rutinemessig beskrevne infeksjoner og identifisere antatte nye prionstammer.
Fordi prioner av bovin spongiform encefalopati (BSE) kan infisere mennesker, kan visse laboratorieprotokoller involvert i arbeidet med BSE kreve bruk av BSL-3-fasiliteter og praksis6. Disse inkluderer bruk av en forseglet sekundær beholder for å transportere potensielle BSE-infiserte vevsprøver i instituttet og laboratoriet. Det inkluderer også utpeking av inneslutningsområder og prion-dedikert utstyr for BSE-forskning og analyse når det er mulig. Dette gjøres for å forhindre forurensning utenfor arbeidsområdet og gi et begrenset rom siden dekontamineringsprosedyrer blir nødvendige.
Følgelig følger laboratoriet for patologi av INIAV anbefalte biosikkerhetsnivå-3 (BSL-3) fasiliteter og praksis6 for å håndtere potensielle prioninfiserte prøver av vev fra storfe, små drøvtyggere og hjortedyr forbundet med TSE-overvåkingen.
Formalinfiksert og parafininnebygd vev som inngår i TSE-diagnostiske eller forskningsprosedyrer, spesielt i sentralnervesystemet, kan være potensielt smittsomt. Derfor må disse faste vevene behandles med maursyre for å redusere smittsomheten til prioner, hvis tilstede, før vevsbehandling. Dette utføres ved å plassere faste, trimmede vev (ca. 2-4 mm tykkelse) i en prosesseringskassett. Kassetten senkes deretter ned i 98% maursyre (i 1 time). Etter nedsenking vaskes kassettene med vev i rennende vann fra springen i 30 minutter, og returneres til fikseringsmiddelet før videre behandling. Dersom vevssnitt ikke behandles før behandling, må postmikrotomiske vevssnitt senkes ned i ufortynnet maursyre i minst 5 minutter før histologisk farging7. IHC-protokollen for PrPSc inkluderer et rutinemessig maursyre-epitop-demasking trinn, som også tjener til å inaktivere prioner7. Etter disse prioninaktiveringstrinnene kan de resulterende faste vevene deretter behandles ved BSL-2 ved hjelp av standard BSL-2-praksis.
Minimumskravet til vevsprøvetaking for TSE-diagnose hos dyr som inngår i TSE-overvåking, er innsamling av hjernestammen (på obexnivå). For å påvise atypisk BSE og skrapesyke anbefales det i tillegg at en del av lillehjernen også samles 1,8. For CWD-diagnose bør både hjernestamme (obex) og retropharyngeale lymfeknuter testes, da PrP Sc kan påvises i lymfoid vev uten påviselig PrPSc i obex9, gjennomgått av Machado et al.10.
Obex-delen av hjernestammen inkluderer diagnostiske TSE-målsteder, nemlig den dorsale vagalkjernen (DVN), ensomhetskjernen (STN) og spinalkanalkjernen i trigeminusnerven (V). Disse områdene presenterer konsekvent bilateral PrPSc-akkumulering , selv i de tidlige stadiene av BSE og klassisk skrapesyke. I kliniske tilfeller av avansert TSE viser alle gråsubstansområdene i hjernestammen utbredt PrPSc-distribusjon 11.
Før snitting og prosessering evalueres hjerneprøvene (figur 1) for å fastslå grad av autolyse og tilstedeværelse av eventuelle vevsskader som potensielt kan svekke prøvens egnethet for IHC-basert bekreftende diagnose8. For å validere integriteten til de forberedende protokollene og analyseresultatene, er TSE-positive og negative vevsprøver inkludert som kontroller i forbindelse med fremstilling av vev fra testtilfeller i hver analyse.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg"> Figur 1: PrPSc IHC-prosedyren. Representasjon som viser trinnvis sekvens av PrPSc IHC-prosedyre fra deparaffinisering av vevsseksjoner til eventuell immunfarging og deteksjon (FFPE - Formalin-fixed paraffin-embedded; Mab - monoklonalt antistoff; DAB - 3,3' diaminobenzidin). Denne figuren ble opprettet i BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">Klikk her for å se en større versjon av denne figuren.</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>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Panreac Applied Chem ITW reagents</td>
			<td>251769</td>
			<td>Undiluted</td>
		</tr>
	</tbody>
</table>

detection of abnormal prion protein by immunohistochemistry

Unormale prionproteiner (PrPSc) er den sykdomsassosierte isoformen av cellulært prionprotein og diagnostiske markører for overførbare spongiforme encefalopatier (TSE). Disse nevrodegenerative sykdommene påvirker mennesker og flere dyrearter og inkluderer skrapesyke, zoonotisk bovin spongiform encefalopati (BSE), kronisk sløsende sykdom av hjortedyr (CWD) og den nylig identifiserte kamelprionsykdommen (CPD). Diagnostisering av TSE er avhengig av immundeteksjon av PrPSc ved bruk av både immunhistokjemi (IHC) og vestlige immunoblotmetoder (WB) på encefalonvev, nemlig hjernestammen (obex-nivå). IHC er en mye brukt metode som bruker primære antistoffer (monoklonal eller polyklonal) mot antigener av interesse i celler i en vevsseksjon. Antistoff-antigenbindingen kan visualiseres ved en fargereaksjon som forblir lokalisert i området av vevet eller cellen der antistoffet ble målrettet. Som sådan, i prionsykdommer, som i andre forskningsfelt, brukes immunhistokjemiteknikkene ikke bare til diagnostiske formål, men også i patogenesestudier. Slike studier innebærer å oppdage PrPSc-mønstre og -typer fra de som tidligere er beskrevet for å identifisere de nye prionstammene. Siden BSE kan infisere mennesker, anbefales det at biosikkerhetslaboratoriet nivå-3 (BSL-3) fasiliteter og / eller praksis brukes til å håndtere storfe, små drøvtyggere og hjortedyrprøver som inngår i TSE-overvåkingen. I tillegg anbefales oppsamlingsutstyr og prion-dedikert utstyr, når det er mulig, for å begrense forurensning. PrPSc IHC-prosedyren består av et maursyre-epitop-demasking trinn som også fungerer som et prioninaktiveringsmål, da formalinfiksert og parafininnebygd vev som brukes i denne teknikken forblir smittsomt. Ved tolkning av resultatene må det utvises forsiktighet for å skille uspesifikk immunmerking fra målmerking. For dette formålet er det viktig å gjenkjenne artefakter av immunmerking oppnådd i kjente TSE-negative kontrolldyr for å skille de fra spesifikke PrP Sc-immunmerkingstyper, som kan variere mellom TSE-stammer, vertsarter og prnp-genotype, nærmere beskrevet her.

Gitt at ikke-spesifikk immunmerking er mulig, er det viktig å fastslå nivået av uspesifikk immunmerking hos kjente TSE-negative kontrolldyr. Dette er et viktig skritt for å tolke spesifikk PrPSc-immunmerking 7 (figur 2) riktig. Ikke-målmerking av anti-PrP-antistoffer har blitt observert å forekomme som diskret intranevronal finpartikkelfarging (tydeligere i hypoglossalkjernen og tilbehørskileatkjernen), uregelmessige finlineære flekktråde...

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

Detection of Abnormal Prion Protein by Immunohistochemistry

Immunmerking av unormalt prionprotein ved bruk av immunhistokjemiske protokoller krever spesifikke prøve- og anti-PrP-antistoffpreparasjonsmetoder. Den nåværende protokollen beskriver de viktigste trinnene i epitopdemasking for å sikre riktig PrP-immunmerking og for å minimere uspesifikk bakgrunnsfarging. Også denne tilnærmingen vurderer biosikkerhetstiltak når man utfører immunhistokjemiske studier med prioninfiserte vev.

Påvisning av unormalt prionprotein ved immunhistokjemi

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

Teknikken tillater bekreftende diagnose av prionsykdommer ved teknikken PrP-skrapesyke i vev, analyse av PrP-skrapetyper og distribusjon for å evaluere straffeavvik fra den kjente PrP-skrapeavsetningsprofilen. Teknikken er standardisert og bruker spesifikke antistoffer mot PrP-skrapesyke, noe som muliggjør visualisering av PrP-skrapesykeavsetningen i vev. Teknikken gir også informasjon om det nevroanatomiske området og typen celler.Begynn med å plassere lysbildene med vevsseksjonene i en fargekurv i rustfritt stål. Dukke opp kurven i et xylenbad. Etter tre minutter, fjern og senk kurven igjen.For å fjerne xylen og rehydrere seksjonene, senk kurven i et absolutt etanolbad. Etter tre minutter, fjern og senk kurven igjen. Lufttørk seksjonene før du legger dem i et 90% etanolbad i ett minutt.Deretter overfører du delen til et 70% etanolbad i et minutt til. For hver etanolbadbehandling, berør glidekurven forsiktig to ganger og tøm kurven før neste overføring. Overfør kurven til et 50% etanolbad i ett minutt.Senk vevsseksjonene forsiktig i 98% maursyre ved romtemperatur i 30 minutter. Skyll seksjonene i vann fra springen i fem minutter etterfulgt av skylling to ganger i destillert vann. Bruk en fargebeholder i rustfritt stål i trykkammeret fylt med 500 ml destillert vann for å forbehandle 10 millimolar citratbuffer ved 98 grader Celsius i 20 minutter.For kvalitetskontrollformål, plasser en del av selvklebende autoklavindikatorbånd på kurven for å overvåke temperatur og trykk. Når alarmen indikerer at utstyret har oppnådd programmets tid og temperatur, senker du kurven med lysbilder. Deretter starter du programmet på trykkammeret til settpunkt 2 og registrerer det første og endelige trykket i dette programmet.For inaktivering av endogen peroksidase, senk glidekurven som inneholder prøveseksjonene i et bad med 3% hydrogenperoksid i metanol i 30 minutter. Bro seksjonene under rennende vann i fem minutter Etter drenering, senk seksjonene i Tris-bufret saltvann i ytterligere fem minutter. For immundeteksjon, plasser hvert lysbilde på en kommersielt tilgjengelig dekselplateholder som er fuktet med Tris-bufret saltvann med vevssiden mot holderen og skyvekanter sammenfallende med de to nedre punktene på holderen.Sørg for å unngå luftbobler. Hold glideholderenheten mellom tommelen og pekefingeren. Hold en finger på toppen av prøvelysbildet og den andre på bunnen av holderen, og plasser deretter enheten i galleriet til systemet.For å sikre at settet er godt montert, fyll brønnen mellom prøvesklien og holderen med Tris-bufret saltvann uten å overfylle den. Fra dette punktet, sørg for at ca 80 mikroliter Tris-bufret saltvann må holdes mellom holderen og lysbildet. Ikke la seksjonene tørke.Reduser bakgrunnsfargingen før behandling med det primære antistoffet ved å pre-inkubere lysbildeprøvene i 30 minutter med 20% normalt serum fra samme art som den sekundære antistoffverten i Tris-bufret saltvann. Uten å vaske seksjonene, påfør 200 mikroliter av den primære antistoffoppløsningen direkte inn i hver brønn i glideholdersettet og inkuber i 60 minutter ved romtemperatur. For å vaske seksjonene, fyll brønnene med Tris-bufret saltvann, og vent i fem minutter.Gjenta vasken to ganger. Deretter fortynnes det biotinylerte sekundære antistoffet ved 1 til 200 konsentrasjon i Tris-bufret saltvann med 10% hesteserum. Reparer ønsket volum avhengig av antall seksjoner som skal behandles.Påfør 200 mikroliter sekundær antistoffløsning på hver brønn i glideholdersettet. Etter 30 minutters inkubasjon ved romtemperatur, gjenta Tris-bufret saltvannsvask. For inkubering med avidin-biotinkompleksperoksidase, lag reagenset 30 minutter før bruk og påfør 200 mikroliter av løsningen i hver brønn i glideplateholderen.Når du er ferdig, inkuber plateholdersettet i 30 minutter ved romtemperatur. Nivået av uspesifikk immunmerking i kjente overførbare spongiforme encefalopatier-negative kontrolldyr ble bestemt for å tolke de spesifikke unormale prionproteinene immunmerking riktig. Ikke-målmerking av anti-PrP-antistoffer ble observert å forekomme som diskret intranevronal finpartikkelfarging, uregelmessige fine lineære tråder av flekk i nevropilen, diffus ikke-partikkelformig merking i noen grå og hvite substansområder og diffus cytoplasmatisk merking av nevroner.Tabellen oppsummerer beskrivelsen av immunmerkede unormale prionproteintyper observert ved bovin spongiform encefalopati, klassisk og atypisk skrapesyke og en skrantesyke hos hjortedyr for positiv diagnose og etablerte kriterier for negative, inkonklusive og uegnede resultater. Alle trinnene er veldig viktige. PH-verdien til løsningene og romtemperaturen er viktige funksjoner for suksessen til denne teknikken.Utvikling av immunhistokjemi for å oppdage både PrP-skrapesyke og type celler kan gi informasjon om hver celle som er involvert i primær patogenese.

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.

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.

Påvisning av Microregional Hypoksi i Mus Cerebral Cortex av to-foton Imaging av endogen 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. ...

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 Immunhistokjemi for å avdekke Complex Brain Topografi

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.

Disseksjon og Immunhistokjemi av larver, pupal og Voksen<em&gt; Drosophila</em&gt; Netthinne

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.

Påvisning av Protein palmitoylation i Cultured hippocampus neurons ved Immunoutfellingsanalyse og 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.

Påvisning av<em&gt; In Situ</em&gt; Protein-protein komplekser på<em&gt; Drosophila</em&gt; Larve Nevromuskulær Junction hjelp Proximity Ligation analysen

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.

Bioluminesens Imaging av nevroinflammasjon i transgene mus etter Peripheral Inokulering av Alfa synukleinfibriller

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.

Overvåking celle til celle overføring av Prion som Protein mengdefunksjoner 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.

Kvantifisere Microglia morfologi Photomicrographs av Immunohistochemistry forberedt Tissue benytter 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 Autoradiographic metode for fastsettelse av regionale priser på cerebral protein syntese 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 ...