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

Questo articolo è stato finanziato dal Progetto POCI-01-0145-FEDER-029947 "Chronic wasting disease risk assessment in Portugal" sostenuto da FCT (Fundação para a Ciência e a Tecnologia) - FEDER -Balcão2020. Inoltre, gli autori dell'unità di ricerca CECAV hanno ricevuto finanziamenti dalla FCT, nell'ambito del progetto UIDB / CVT / 0772 / 2020.Ringraziamo Bruce C. Campbell, direttore della ricerca (in pensione), Western Regional Research Center, USDA, per la sua assistenza.

La procedura IHC qui descritta è una componente del progetto di ricerca FAIRJ-CT98-7021, "The establishment of a European network for the surveillance of ruminant TSE and the standardization and harmonization of the process and criteria for the identification of suspect cases". Segue i criteri diagnostici definiti dall'Organizzazione mondiale per la salute animale, WOAH (precedentemente denominato Office International des Épizooties, OIE)1 e European Reference Laboratory for animal TSE 8,11...

<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>

Le TSE sono potenziali malattie zoonotiche. Dopo la comparsa della BSE nel 1986 nel Regno Unito, il Portogallo è diventato uno degli Stati membri dell'Unione europea con una maggiore incidenza di questa malattia14,15. Al fine di controllare questa malattia, altre TSE che sono emerse (scrapie classica e atipica, varianti della BSE, e attualmente la sorveglianza della malattia del deperimento cronico nei cervidi), meccanismi di sorveglianza sono stati sviluppati d...

Secondo l'ipotesi prionica, l'isoforma anomala (PrPSc) è l'unico componente primario dell'agente infettivo nelle encefalopatie spongiformi trasmissibili (TSE). La conferma per la diagnosi di TSE si basa sull'immunorilevazione della PrPSc mediante l'applicazione di protocolli immunoistochimici (IHC) e/o metodi immunoblot occidentali (WB) dei tessuti dell'encefalo1.
L'IHC è un metodo che impiega anticorpi monoclonali o, in alcuni casi, policlonali (come anticorpi primari) come primo passo nell'immunocolorazione di antigeni di interesse specificamente mirati situati nelle cellule di una sezione tissutale. Qualsiasi legame efficace anticorpo-antigene primario viene quindi visualizzato utilizzando anticorpi secondari specifici per gli anticorpi primari. Questi anticorpi secondari sono coniugati agli enzimi, come la perossidasi di rafano (HRP) o la fosfatasi alcalina (AP). La visualizzazione si ottiene quindi aggiungendo un substrato a questi enzimi, producendo prodotti di colore insolubili localizzati in aree in cui gli anticorpi primari sono legati agli antigeni bersaglio. Una migliore visualizzazione può essere ottenuta contrastando, in cui un colorante viene utilizzato per creare un contrasto tra tessuto immunomarcato e non immunomarcato2.
Con IHC che utilizza tessuti incorporati in paraffina fissati in formalina (FFPE), la fissazione della formalina può annullare l'efficacia degli anticorpi primari a causa della reticolazione mediante formaldeide e riscaldamento e disidratazione durante l'incorporazione di paraffina. Questi modificano la conformazione delle proteine, distruggendo, denaturando o mascherando gli epitopi, diminuendo o abrogando così la loro rilevazione3. Come tale, ciò richiede il recupero dell'antigene (AR). Le tecniche AR interrompono il cross-linking dei gruppi chimici correlati alla formaldeide nelle molecole antigeniche, ripristinando o smascherando così la conformazione originale antigene-proteina. Ciò si traduce nel recupero dell'affinità anticorpo-antigene (epitopo) per l'immunomarcatura. L'eventuale efficacia dell'AR dipende dalle qualità dell'antigene bersaglio e/o dell'anticorpo primario2.
Il recupero dell'antigene indotto dal calore (epitopo) (HIER) è una procedura di AR3 e viene utilizzato di routine per il rilevamento di PrPSc IHC, come descritto nel presente documento. L'IHC è essenziale per la diagnosi e viene utilizzato nei laboratori di ricerca per determinare la distribuzione tissutale di un antigene associato alla patologia. È ampiamente usato nella diagnosi e nella ricerca di cancro, neuroscienze e malattie infettive4, tra gli altri. Per le TSE, l'IHC svolge un ruolo importante nella diagnosi e nella ricerca per confermare e studiare la distribuzione di PrPSc negli ospiti naturali e nei modelli sperimentali. IHC contribuisce agli studi sulla patogenesi prionica e all'analisi dei tipi e dei modelli di deposizione di PrPSc , vale a dire, nei tessuti neurali5, per rilevare deviazioni dalle infezioni descritte di routine e identificare nuovi ceppi prionici putativi.
Poiché i prioni dell'encefalopatia spongiforme bovina (BSE) possono infettare l'uomo, alcuni protocolli di laboratorio coinvolti nel lavoro con la BSE possono richiedere l'uso di strutture e pratiche BSL-36. Questi includono l'uso di un contenitore secondario sigillato per trasportare potenziali campioni di tessuto infetto da BSE all'interno dell'istituto e del laboratorio. Comprende anche la designazione di aree di contenimento e attrezzature dedicate ai prioni per la ricerca e l'analisi della BSE ogniqualvolta possibile. Questo viene fatto per prevenire la contaminazione al di fuori dell'area di lavoro e fornire uno spazio ristretto poiché le procedure di decontaminazione diventano necessarie.
Di conseguenza, il Laboratorio di Patologia dell'INIAV segue le strutture e le pratiche raccomandate di livello 3 di biosicurezza (BSL-3)6 per gestire potenziali campioni di tessuti infetti da prioni di bovini, piccoli ruminanti e cervidi associati alla sorveglianza delle TSE.
I tessuti fissati in formalina e incorporati in paraffina inclusi nelle procedure diagnostiche o di ricerca sulle TSE, specialmente nel sistema nervoso centrale, possono essere potenzialmente infettivi. Quindi, questi tessuti fissi devono essere trattati con acido formico per ridurre l'infettività dei prioni, se presenti, prima della lavorazione dei tessuti. Questo viene eseguito posizionando tessuti fissi e rifilati (circa 2-4 mm di spessore) in una cassetta di lavorazione. La cassetta viene quindi immersa in acido formico al 98% (per 1 ora). Dopo l'immersione, le cassette con i fazzoletti vengono lavate in acqua corrente del rubinetto per 30 minuti e restituite al fissativo prima di ulteriori elaborazioni. Se le sezioni di tessuto non vengono trattate prima della lavorazione, le sezioni di tessuto post-microtomico devono essere immerse in acido formico non diluito per almeno 5 minuti prima della colorazione istologica7. Il protocollo IHC per PrPSc include una fase di routine di epitopo dell'acido formico, che serve anche a inattivare i prioni7. Dopo queste fasi di inattivazione dei prioni, i tessuti fissi risultanti possono quindi essere elaborati a BSL-2 utilizzando pratiche standard BSL-2.
Il requisito minimo di campionamento dei tessuti per la diagnosi di TSE in qualsiasi animale incluso nella sorveglianza delle TSE è la raccolta del tronco encefalico (a livello dell'obex). Inoltre, per rilevare BSE e scrapie atipiche, si consiglia di raccogliere anche una parte del cervelletto 1,8. Per la diagnosi di CWD, sia il tronco encefalico (obex) che i linfonodi retrofaringei dovrebbero essere testati poiché PrP Sc potrebbe essere rilevato nei tessuti linfoidi senza PrPSc rilevabile nell'obex9, esaminato da Machado et al.10.
La porzione obex del tronco encefalico comprende siti bersaglio diagnostici TSE, vale a dire il nucleo vagale dorsale (DVN), il nucleo del tratto solitario (STN) e il nucleo del tratto spinale del nervo trigemino (V). Queste aree presentano costantemente un accumulo bilaterale di PrPSc , anche nelle prime fasi della BSE e della scrapie classica. Nei casi clinici di TSE avanzata, tutte le aree della materia grigia all'interno del tronco encefalico mostrano una diffusa distribuzione di PrPSc 11.
Prima del sezionamento e dell'elaborazione, i campioni cerebrali vengono valutati (Figura 1) per accertare il livello di autolisi e la presenza di eventuali danni tissutali che potrebbero compromettere l'idoneità del campione per la diagnosi di conferma basata su IHC8. Per convalidare l'integrità dei protocolli preparativi e dei risultati analitici, i campioni di tessuto positivo e negativo alla TSE sono inclusi come controlli in combinazione con la preparazione dei tessuti dai casi di prova in ciascun test.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg"> Figura 1: La procedura PrPSc IHC. Rappresentazione che mostra la sequenza passo-passo della procedura PrPSc IHC dalla deparaffinizzazione di sezioni tissutali all'eventuale immunocolorazione e rilevazione (FFPE - Formalin-fixed paraffin-embedded; Mab - anticorpo monoclonale; DAB - 3,3' diaminobenzidina). Questa figura è stata creata nel BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">Fare clic qui per visualizzare una versione ingrandita di questa figura.</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

Le proteine prioniche anomale (PrPSc) sono l'isoforma associata alla malattia della proteina prionica cellulare e i marcatori diagnostici delle encefalopatie spongiformi trasmissibili (TSE). Queste malattie neurodegenerative colpiscono l'uomo e diverse specie animali e comprendono la scrapie, l'encefalopatia spongiforme bovina zoonotica (BSE), la malattia del deperimento cronico dei cervidi (CWD) e la malattia da prioni del cammello (CPD) recentemente identificata. La diagnosi di TSE si basa sull'immunorilevazione della PrPSc mediante l'applicazione sia dell'immunoistochimica (IHC) che dei metodi immunoblot occidentali (WB) sui tessuti dell'encefalo, vale a dire il tronco cerebrale (livello obex). IHC è un metodo ampiamente utilizzato che utilizza anticorpi primari (monoclonali o policlonali) contro antigeni di interesse nelle cellule di una sezione tissutale. Il legame anticorpo-antigene può essere visualizzato da una reazione di colore che rimane localizzata nell'area del tessuto o della cellula in cui l'anticorpo è stato preso di mira. Pertanto, nelle malattie da prioni, come in altri campi di ricerca, le tecniche di immunoistochimica non sono utilizzate solo per scopi diagnostici, ma anche negli studi di patogenesi. Tali studi prevedono la rilevazione dei modelli e dei tipi di PrPSc da quelli precedentemente descritti per identificare i nuovi ceppi prionici. Poiché la BSE può infettare l'uomo, si raccomanda di utilizzare strutture e/o pratiche di laboratorio di biosicurezza di livello 3 (BSL-3) per manipolare campioni di bovini, piccoli ruminanti e cervidi inclusi nella sorveglianza delle TSE. Inoltre, si raccomandano attrezzature di contenimento e dedicate ai prioni, quando possibile, per limitare la contaminazione. La procedura PrPSc IHC consiste in una fase di epitopo dell'acido formico che agisce anche come misura di inattivazione dei prioni, poiché i tessuti fissati in formalina e incorporati in paraffina utilizzati in questa tecnica rimangono infettivi. Nell'interpretare i risultati, è necessario prestare attenzione a distinguere l'immunomarcatura non specifica dall'etichettatura target. A tal fine, è importante riconoscere gli artefatti dell'immunomarcatura ottenuti in animali di controllo TSE-negativi noti per differenziarli da specifici tipi di immunomarcatura PrPSc , che possono variare tra ceppi di TSE, specie ospiti e genotipo prnp , ulteriormente descritti nel presente documento.

Dato che è possibile una marcatura immunomarcatrice bersaglio non specifica, è importante accertare il livello di immunomarcatura non specifica negli animali di controllo noti TSE-negativi. Questo è un passo importante per interpretare correttamente specifici immunolabeling PrPSc 7 (Figura 2). È stato notato che la marcatura non bersaglio da parte di anticorpi anti-PrP si verifica come colorazione intraneuronale discreta del particolato fine (...

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

Detection of Abnormal Prion Protein by Immunohistochemistry

L'immunomarcatura della proteina prionica anomala utilizzando protocolli immunoistochimici richiede specifiche metodologie di preparazione di campioni e anticorpi anti-PrP. Il presente protocollo descrive i passaggi chiave del demasking degli epitopi per garantire una corretta etichettatura immunologica della PrP e per ridurre al minimo la colorazione di fondo non specifica. Inoltre, questo approccio considera le misure di biosicurezza quando si conducono studi di immunoistochimica con i tessuti infetti da prioni.

Rilevazione di proteine prioniche anomale mediante immunoistochimica

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

La tecnica consente la diagnosi di conferma delle malattie da prioni mediante la tecnica della scrapie PrP nei tessuti, analizzando i tipi di scrapie PrP e la distribuzione per valutare la deviazione punitiva al profilo noto di deposizione della scrapie PrP. La tecnica è standardizzata e utilizza anticorpi specifici contro la scrapie PrP, consentendo la visualizzazione della deposizione di scrapie PrP nei tessuti. La tecnica fornisce anche informazioni sull'area neuroanatomica e sul tipo di cellule.Inizia posizionando i vetrini con le sezioni di tessuto in un cestello colorante in acciaio inossidabile. Far emergere il cesto in un bagno di xilene. Dopo tre minuti, rimuovere e immergere nuovamente il cestello.Per rimuovere lo xilene e reidratare le sezioni, immergere il cestello in un bagno di etanolo assoluto. Dopo tre minuti, rimuovere e immergere nuovamente il cestello. Asciugare all'aria le sezioni prima di metterle in un bagno di etanolo al 90% per un minuto.Quindi, trasferire la sezione in un bagno di etanolo al 70% per un altro minuto. Per ogni trattamento con bagno di etanolo, agitare delicatamente il cestello di scorrimento due volte e scolare il cestello prima del trasferimento successivo. Trasferire il cestello in un bagno di etanolo al 50% per un minuto.Immergere accuratamente le sezioni di tessuto in acido formico al 98% a temperatura ambiente per 30 minuti. Risciacquare le sezioni in acqua di rubinetto per cinque minuti, quindi risciacquare due volte in acqua distillata. Utilizzare un contenitore di colorazione in acciaio inossidabile nella camera a pressione riempito con 500 millilitri di acqua distillata per pretrattare il tampone citrato da 10 millimolari a 98 gradi Celsius per 20 minuti.Ai fini del controllo qualità, posizionare una sezione di nastro adesivo per autoclave sul cestello per monitorare la temperatura e la pressione. Una volta che l'allarme indica che l'apparecchiatura ha raggiunto il tempo e la temperatura del programma, immergere il cestello con le diapositive. Quindi, avviare il programma sulla camera di pressione per impostare il punto 2 e registrare la pressione iniziale e finale di questo programma.Per l'inattivazione della perossidasi endogena, immergere il cestello del vetrino contenente le sezioni del campione in un bagno di perossido di idrogeno al 3% in metanolo per 30 minuti. Colmare le sezioni sotto l'acqua corrente per cinque minuti Dopo il drenaggio, immergere le sezioni in soluzione salina tamponata Tris per altri cinque minuti. Per il rilevamento immunologico, posizionare ogni vetrino su un supporto per piastra di copertura disponibile in commercio pre-inumidito con soluzione salina tamponata Tris con il lato del tessuto rivolto verso il supporto e i bordi del vetrino coincidenti con i due punti inferiori del supporto.Assicurarsi di evitare bolle d'aria. Tenere il gruppo portaslitta tra il pollice e l'indice. Tenere un dito sulla parte superiore del vetrino campione e l'altro sul fondo del supporto, quindi posizionare l'assieme nella galleria del sistema.Per garantire che il set sia ben assemblato, riempire il pozzetto tra il vetrino del campione e il supporto con soluzione salina tamponata Tris senza traboccare. Da questo punto, assicurarsi che circa 80 microlitri di soluzione salina tamponata Tris debbano essere trattenuti tra il supporto e il vetrino. Non lasciare asciugare le sezioni.Ridurre la colorazione di fondo prima del trattamento con l'anticorpo primario pre-incubando i campioni di vetrino per 30 minuti con il 20% di siero normale della stessa specie dell'ospite anticorpale secondario nella soluzione salina tamponata Tris. Senza lavare le sezioni, applicare 200 microlitri della soluzione anticorpale primaria direttamente in ciascun pozzetto del set portavetrino e incubare per 60 minuti a temperatura ambiente. Per lavare le sezioni, riempire i pozzetti con soluzione salina tamponata Tris e attendere cinque minuti.Ripetere il lavaggio due volte. Quindi, diluire l'anticorpo secondario biotinilato a concentrazione da 1 a 200 in soluzione salina tamponata Tris con siero di cavallo al 10%. Riparare il volume richiesto in base al numero di sezioni da trattare.Applicare 200 microlitri di soluzione anticorpale secondaria a ciascun pozzetto del set di portavetrini. Dopo 30 minuti di incubazione a temperatura ambiente, ripetere il lavaggio salino Tris-buffered. Per l'incubazione con perossidasi del complesso avidina-biotina, preparare il reagente 30 minuti prima dell'uso e applicare 200 microlitri della soluzione in ciascun pozzetto del supporto della piastra di scorrimento.Una volta fatto, incubare il portapiastre impostato per 30 minuti a temperatura ambiente. Il livello di immunomarcatura non specifica in animali di controllo negativi alle encefalopatie spongiformi trasmissibili note è stato determinato per interpretare correttamente l'immunomarcatura delle proteine prioniche anomale specifiche. È stato notato che la marcatura non bersaglio da parte di anticorpi anti-PrP si verifica come colorazione intraneuronale discreta del particolato fine, fili lineari irregolari di colorazione nel neuropil, marcatura diffusa non particolata in alcune aree della sostanza grigia e bianca e marcatura citoplasmatica diffusa dei neuroni.La tabella riassume la descrizione dei tipi di proteine prioniche anomale immunomarcate osservati nell'encefalopatia spongiforme bovina, nella scrapie classica e atipica e in una malattia da deperimento cronico dei cervidi per una diagnosi positiva e criteri stabiliti per risultati negativi, inconcludenti e inadatti. Tutti i passaggi sono molto importanti. Il pH delle soluzioni e la temperatura ambiente sono caratteristiche importanti per il successo di questa tecnica.Lo sviluppo di immunoistochimica per rilevare sia la scrapie PrP che il tipo di cellule può fornire informazioni su ciascuna cellula coinvolta nella patogenesi primaria.

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.

La combinazione di coloranti lipofili, In situ Ibridazione, Immunoistochimica e Istologia

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

Ricerca

Neuroscienze

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.

Rilevamento di ipossia microregionale nella corteccia cerebrale di topo Imaging a due fotoni di fluorescenza endogena NADH

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.

Immunoistochimica Wholemount per rivelare Topografia Cervello Complex

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.

Dissezione e immunoistochimica di larvale, pupa e adulto<em&gt; Drosophila</em&gt; Retine

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.

Rilevamento di palmitoilazione proteina in colture di neuroni ippocampali di immunoprecipitazione e di acil-Biotina 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.

Rilevamento di<em&gt; In Situ</em&gt; Complessi proteina-proteina del<em&gt; Drosophila</em&gt; Larvale neuromuscolare Junction Utilizzando prossimità legatura Assay

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

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

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

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

Bioluminescenza Imaging di Neuroinflammation in topi transgenici Dopo periferica inoculazione di alfa-sinucleina fibrille

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.

Monitoraggio-cellula-trasmissione degli aggregati della proteina del Prion-come in 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.

Quantificare la morfologia di Microglia da microfotografie di Immunohistochemistry preparato tessuto usando 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.

Metodo autoradiografico quantitativo per la determinazione dei tassi regionali di sintesi proteica cerebrale 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 ...