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 artikel blev finansieret af projektet POCI-01-0145-FEDER-029947 "Chronic wasting disease risk assessment in Portugal" støttet af FCT (Fundação para a Ciência e a Tecnologia) - FEDER -Balcão2020. Også forfatterne af forskningsenheden CECAV modtog finansiering fra FCT under projektet UIDB / CVT / 0772/2020.Vi takker Bruce C. Campbell, forskningsdirektør (pensioneret), Western Regional Research Center, USDA, for hans hjælp.

IHC-proceduren, der er beskrevet heri, er en del af forskningsprojektet FAIRJ-CT98-7021, "Etablering af et europæisk netværk til overvågning af TSE hos drøvtyggere og standardisering og harmonisering af processen og kriterierne for identifikation af mistænkte tilfælde". Det følger de diagnosekriterier, der er fastlagt af Verdensorganisationen for Dyresundhed, WOAH (tidligere benævnt Det Internationale Kontor for Epizootier, OIE)1 og Det Europæiske Referencelaboratorium for TSE hos Dyr<sup...

<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 potentielle zoonotiske sygdomme. Efter fremkomsten af BSE i 1986 i Det Forenede Kongerige blev Portugal en af de EU-medlemsstater, der havde en højere forekomst af denne sygdom14,15. For at bekæmpe denne sygdom er der udviklet andre TSE'er (klassisk og atypisk scrapie, BSE-varianter og i øjeblikket overvågning af chronic wasting disease hos hjortedyr), overvågningsmekanismer af Generaldirektoratet for Fødevarer og Veterinær (DGAV) og National Instit...

Ifølge prionhypotesen er den abnorme isoform (PrPSc) den primære eller eneste komponent i det infektiøse agens i transmissible spongiforme encephalopatier (TSE'er). Bekræftelse til diagnosticering af TSE afhænger af immunpåvisning af PrPSc ved anvendelse af immunhistokemiske (IHC) protokoller og/eller vestlige immunblotmetoder (WB) af encefalonvæv1.
IHC er en metode, der anvender monoklonale eller i nogle tilfælde polyklonale antistoffer (som primære antistoffer) som et første trin i immunfarvning af specifikt målrettede antigener af interesse placeret i celler i en vævssektion. Enhver effektiv primær antistof-antigenbinding visualiseres derefter ved hjælp af sekundære antistoffer, der er specifikke for de primære antistoffer. Disse sekundære antistoffer konjugeres til enzymer, såsom peberrodsperoxidase (HRP) eller alkalisk fosfatase (AP). Visualisering opnås derefter ved at tilføje et substrat til disse enzymer, der producerer uopløselige farveprodukter lokaliseret i områder, hvor de primære antistoffer er bundet til de målrettede antigener. Forbedret visualisering kan opnås ved modfarvning, hvor et farvestof bruges til at skabe en kontrast mellem immunmærket og ikke-immunmærket væv2.
Ved IHC ved anvendelse af formalinfikseret paraffinindlejret væv (FFPE) kan formalinfiksering ophæve effektiviteten af primære antistoffer på grund af tværbinding med formaldehyd og opvarmning og dehydrering under paraffinindlejring. Disse ændrer konformationen af proteiner, ødelægger, denaturerer eller maskerer epitoperne og mindsker eller ophæver dermed deres påvisning3. Som sådan kræver dette antigenhentning (AR). AR-teknikkerne forstyrrer formaldehydrelateret kemisk gruppetværbinding i de antigene molekyler og genopretter eller afslører således den oprindelige antigen-proteinkonformation. Dette resulterer i at genvinde antistof-antigen (epitop) affinitet til immunmærkning. Den endelige effekt af AR afhænger af kvaliteten af det målrettede antigen og / eller det primære antistof2.
Varmeinduceret antigen (epitop) hentning (HIER) er en procedure i AR3 og anvendes rutinemæssigt til PrPSc IHC-detektion, som beskrevet heri. IHC er afgørende for diagnose og anvendes i forskningslaboratorier til at bestemme vævsfordelingen af et patologiassocieret antigen. Det bruges i vid udstrækning til diagnosticering og forskning i kræft, neurovidenskab og infektionssygdomme4, blandt andre. For TSE spiller IHC en vigtig rolle i diagnosticering og forskning til bekræftelse og undersøgelse af PrPSc-fordeling i naturlige værter og eksperimentelle modeller. IHC bidrager til prionpatogeneseundersøgelser og analyse af PrP Sc-aflejringstyper og -mønstre, nemlig i neurale væv5, for at påvise afvigelser fra rutinemæssigt beskrevne infektioner og identificere formodede nye prionstammer.
Da prioner af bovin spongiform encephalopati (BSE) kan inficere mennesker, kan visse laboratorieprotokoller, der er involveret i arbejdet med BSE, kræve anvendelse af BSL-3-faciliteter og -praksis6. Disse omfatter anvendelse af en forseglet sekundær beholder til transport af potentielle BSE-inficerede vævsprøver inden for instituttet og laboratoriet. Det omfatter også udpegelse af indeslutningsområder og prionsærligt udstyr til BSE-forskning og -analyse, når det er muligt. Dette gøres for at forhindre kontaminering uden for arbejdsområdet og give et begrænset rum, da dekontamineringsprocedurer bliver nødvendige.
I overensstemmelse hermed følger laboratoriet for patologi i INIAV anbefalede faciliteter og praksis for biosikkerhedsniveau-3 (BSL-3)6 for at håndtere potentielle prioninficerede prøver af væv fra kvæg, små drøvtyggere og hjortedyr, der er forbundet med TSE-overvågningen.
Formalinfikseret og paraffinindlejret væv, der indgår i TSE-diagnosticerings- eller forskningsprocedurer, især i centralnervesystemet, kan være potentielt smitsomt. Derfor skal disse faste væv behandles med myresyre for at reducere smitsomheden af prioner, hvis de er til stede, før vævsbehandling. Dette udføres ved at placere faste, trimmede væv (ca. 2-4 mm tykkelse) i en behandlingskassette. Kassetten nedsænkes derefter i 98% myresyre (i 1 time). Efter nedsænkning vaskes kassetterne med væv i rindende ledningsvand i 30 minutter og returneres til fikseringsmidlet inden videre behandling. Hvis vævssektioner ikke behandles før behandling, skal postmikrotomiske vævssektioner nedsænkes i ufortyndet myresyre i mindst 5 minutter før histologisk farvning7. IHC-protokollen for PrPSc inkluderer et rutinemæssigt myresyreepitop-demmaskeringstrin, der også tjener til at inaktivere prioner7. Efter disse prioninaktiveringstrin kan det resulterende faste væv derefter behandles ved BSL-2 ved hjælp af standard BSL-2-praksis.
Minimumskravet til vævsprøvetagning til TSE-diagnosticering hos alle dyr, der er omfattet af TSE-overvågningen, er indsamling af hjernestammen (på obex-niveau). For at påvise atypisk BSE og scrapie anbefales det desuden, at en del af lillehjernen også indsamles 1,8. Til CWD-diagnose bør både hjernestamme (obex) og retropharyngeale lymfeknuder testes, da PrP Sc kunne påvises i lymfoide væv uden påviselig PrPSc i obex9, gennemgået af Machado et al.10.
Den obex-del af hjernestammen omfatter diagnostiske TSE-målsteder, nemlig dorsal vagalkernen (DVN), ensomhedskanalkernen (STN) og rygmarvskernen i trigeminusnerven (V). Disse områder udviser konsekvent bilateral akkumulering af PrPSc , selv i de tidlige stadier af BSE og klassisk scrapie. I kliniske tilfælde af fremskreden TSE viser alle gråstofområder i hjernestammen udbredt PrPSc-fordeling 11.
Før sektionering og behandling evalueres hjerneprøverne (figur 1) for at fastslå autolyseniveauet og tilstedeværelsen af eventuelle vævsskader, der potentielt kan kompromittere prøvens egnethed til IHC-baseret bekræftende diagnose8. For at validere integriteten af de præparative protokoller og analyseresultaterne indgår TSE-positive og negative vævsprøver som kontroller i forbindelse med fremstilling af væv fra testtilfælde i hvert assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg"> Figur 1: PrPSc IHC-proceduren. Repræsentation, der viser den trinvise sekvens af PrPSc IHC-proceduren fra deparaffinisering af vævssektioner til eventuel immunfarvning og detektion (FFPE - Formalin-fixed paraffin-embedded; Mab - monoklonalt antistof; DAB - 3,3' diaminobenzidin). Denne figur blev oprettet i BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">Klik her for at se en større version af denne 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>
		</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 sygdomsassocierede isoform af cellulært prionprotein og diagnostiske markører for transmissible spongiforme encephalopatier (TSE'er). Disse neurodegenerative sygdomme påvirker mennesker og flere dyrearter og omfatter scrapie, zoonotisk bovin spongiform encephalopati (BSE), chronic wasting disease hos hjortedyr (CWD) og den nyligt identificerede kamelprionsygdom (CPD). Diagnosticering af TSE er afhængig af immunpåvisning af PrPSc ved anvendelse af både immunhistokemi (IHC) og vestlige immunoblotmetoder (WB) på encefalonvæv, nemlig hjernestammen (obex-niveau). IHC er en meget anvendt metode, der bruger primære antistoffer (monoklonale eller polyklonale) mod antigener af interesse i celler i en vævssektion. Antistof-antigenbindingen kan visualiseres ved en farvereaktion, der forbliver lokaliseret i det område af vævet eller cellen, hvor antistoffet var målrettet. Som sådan anvendes immunhistokemiske teknikker i prionsygdomme som inden for andre forskningsområder ikke kun til diagnostiske formål, men også i patogeneseundersøgelser. Sådanne undersøgelser involverer påvisning af PrPSc-mønstre og -typer fra dem, der tidligere er beskrevet, for at identificere de nye prionstammer. Da BSE kan inficere mennesker, anbefales det, at faciliteter og/eller praksis på biosikkerhedslaboratorieniveau 3 (BSL-3) anvendes til håndtering af kvæg, små drøvtyggere og hjortedyrprøver, der indgår i TSE-overvågningen. Derudover anbefales indeslutnings- og priondedikeret udstyr, når det er muligt, for at begrænse kontaminering. PrPSc IHC-proceduren består af et myresyreepitop-demmaskeringstrin, der også fungerer som en prioninaktiveringsforanstaltning, da formalinfikseret og paraffinindlejret væv, der anvendes i denne teknik, forbliver smitsomt. Ved fortolkning af resultaterne skal man sørge for at skelne ikke-specifik immunmærkning fra målmærkning. Til dette formål er det vigtigt at genkende artefakter af immunmærkning opnået i kendte TSE-negative kontroldyr for at skelne dem fra specifikke PrP Sc-immunmærkningstyper, som kan variere mellem TSE-stammer, værtsarter og prnp-genotype, yderligere beskrevet heri.

Da ikke-specifik målimmunmærkning er mulig, er det vigtigt at fastslå niveauet for ikke-specifik immunmærkning hos kendte TSE-negative kontroldyr. Dette er et vigtigt skridt til korrekt fortolkning af specifik PrPSc-immunmærkning 7 (figur 2). Ikke-målmærkning med anti-PrP-antistoffer er blevet bemærket at forekomme som diskret intraneuronal finpartikelfarvning (mere tydelig i hypoglossalkernen og tilbehørscuneatkernen), uregelmæssige fin...

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

Detection of Abnormal Prion Protein by Immunohistochemistry

Immunmærkning af unormalt prionprotein ved hjælp af immunhistokemiske protokoller kræver specifikke prøve- og anti-PrP-antistofpræparationsmetoder. Denne protokol beskriver de vigtigste trin i epitopmaskering for at sikre korrekt PrP-immunmærkning og for at minimere ikke-specifik baggrundsfarvning. Denne tilgang overvejer også biosikkerhedsforanstaltninger ved udførelse af immunhistokemiske undersøgelser med de prioninficerede væv.

Påvisning af unormalt prionprotein ved immunhistokemi

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

Teknikken tillader bekræftende diagnose af prionsygdomme ved hjælp af teknikken PrP-scrapie i væv, analyse af PrP-scrapietyper og fordeling for at evaluere straffeafvigelse til den kendte PrP-scrapieaflejringsprofil. Teknikken er standardiseret og bruger specifikke antistoffer mod PrP-scrapie, hvilket muliggør visualisering af PrP-scrapieaflejring i væv. Teknikken giver også information om det neuroanatomiske område og typen af celler.Begynd med at placere diasene med vævssektionerne i en rustfri stålfarvekurv. Gå kurven ud i et xylenbad. Efter tre minutter fjernes kurven og nedsænkes igen.For at fjerne xylen og rehydrere sektionerne nedsænkes kurven i et absolut ethanolbad. Efter tre minutter fjernes kurven og nedsænkes igen. Lufttør sektionerne, før du lægger dem i et 90% ethanolbad i et minut.Overfør derefter sektionen til et 70% ethanolbad i endnu et minut. For hver ethanolbadbehandling omrøres glidekurven forsigtigt to gange og tømmes kurven inden næste overførsel. Overfør kurven til et 50% ethanolbad i et minut.Nedsænk forsigtigt vævssektionerne i 98% myresyre ved stuetemperatur i 30 minutter. Skyl sektionerne i ledningsvand i fem minutter efterfulgt af skylning to gange i destilleret vand. Brug en farvningsbeholder i rustfrit stål i trykkammeret fyldt med 500 ml destilleret vand til at forbehandle 10 millimolær citratbuffer ved 98 grader Celsius i 20 minutter.Til kvalitetskontrolformål skal du placere et afsnit af klæbende autoklaveindikatortape på kurven for at overvåge temperatur og tryk. Når alarmen angiver, at udstyret har nået programtid og temperatur, skal du nedsænke kurven med dias. Start derefter programmet på trykkammeret til sætpunkt 2 og registrer det indledende og endelige tryk i dette program.Til inaktivering af endogen peroxidase nedsænkes diaskurven med prøvesektionerne i et bad med 3% hydrogenperoxid i methanol i 30 minutter. Bro sektionerne under rindende vand i fem minutter Efter dræning nedsænkes sektionerne i Tris-bufret saltvand i yderligere fem minutter. Til immundetektion anbringes hvert objektglas på en kommercielt tilgængelig dækpladeholder, der er fugtet med Tris-bufret saltvand, hvor vævssiden vender mod holderen og glidekanterne falder sammen med holderens to nederste punkter.Sørg for at undgå luftbobler. Hold lysbilledholderenheden mellem tommel- og pegefinger. Hold den ene finger oven på prøveglasset og den anden i bunden af holderen, og placer derefter enheden i systemets galleri.For at sikre, at sættet er godt samlet, skal du fylde brønden mellem prøveglasset og holderen med Tris-bufret saltvand uden at flyde over det. Fra dette punkt skal du sikre dig, at ca. 80 mikroliter Tris-bufret saltvand skal bevares mellem holderen og objektglasset. Lad ikke sektionerne tørre.Reducer baggrundsfarvningen før behandlingen med det primære antistof ved at præinkubere diasprøverne i 30 minutter med 20% normalt serum fra samme art som den sekundære antistofvært i Tris-bufret saltvand. Uden at vaske sektionerne påføres 200 mikroliter af den primære antistofopløsning direkte i hvert hul i diasholdersættet og inkuberes i 60 minutter ved stuetemperatur. For at vaske sektionerne skal du fylde brøndene med Tris-bufret saltvand og vente i fem minutter.Gentag vasken to gange. Fortynd derefter det biotinylerede sekundære antistof ved 1 til 200 koncentration i Tris-bufret saltvand med 10% hesteserum. Reparer det krævede volumen afhængigt af antallet af sektioner, der skal behandles.Påfør 200 mikroliter sekundær antistofopløsning på hver brønd i diasholdersættet. Efter 30 minutters inkubation ved stuetemperatur gentages Tris-bufret saltvandsvask. Til inkubation med avidin-biotinkompleks peroxidase fremstilles reagenset 30 minutter før brug og påføres 200 mikroliter af opløsningen i hvert hul i glidepladeholderen.Når det er gjort, inkuberes pladeholderen i 30 minutter ved stuetemperatur. Niveauet af ikke-specifik immunmærkning hos kendte transmissible spongiforme encephalopatier-negative kontroldyr blev bestemt til at fortolke immunmærkningen af de specifikke abnorme prionproteiner korrekt. Ikke-målmærkning ved anti-PrP-antistoffer blev bemærket at forekomme som diskret intraneuronal finpartikelfarvning, uregelmæssige fine lineære tråde af plet i neuropilen, diffus ikke-partikelmærkning i nogle grå og hvide stofområder og diffus cytoplasmatisk mærkning af neuroner.Tabellen opsummerer beskrivelsen af immunmærkede abnorme prionproteintyper observeret i bovin spongiform encephalopati, klassisk og atypisk scrapie og en chronic wasting disease hos hjortedyr til en positiv diagnose og etablerede kriterier for negative, inkonklusive og uegnede resultater. Alle trin er meget vigtige. Opløsningernes pH og stuetemperaturen er vigtige træk for succesen med denne teknik.Udvikling af immunhistokemi til påvisning af både PrP-scrapie og celletype kan give information om hver celle, der er involveret i primepatogenese.

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 af Microregional Hypoxia i mus hjernebarken ved to-foton Imaging af endogent 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 Immunhistokemi for at afsløre 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.

Dissektion og Immunhistokemi af Larvernes, Pupal og Voksen<em&gt; Drosophila</em&gt; Nethinder

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 af protein palmitoylering i dyrkede hippocampale neuroner ved immunopræcipitation 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 af<em&gt; In Situ</em&gt; Protein-protein-komplekser på<em&gt; Drosophila</em&gt; Larve Neuromuskulær Junction Brug Proximity ligeringsassayet

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.

Bioluminescens Billeddannelse af Neuroinflammation i transgene mus efter perifer Inokulering af alfa-synuclein 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.

Overvågning celle til celle Transmission af Prion-lignende Protein aggregater 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.

Kvantificere mikroglia morfologi fra mikrofotografier af Immunhistokemi forberedt væv ved hjælp af 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 metode til bestemmelse af de regionale rater for 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 ...