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

Este artículo fue financiado por el Proyecto POCI-01-0145-FEDER-029947 "Evaluación del riesgo de desgaste crónico en Portugal" apoyado por FCT (Fundação para a Ciência e a Tecnologia) - FEDER -Balcão2020. Asimismo, los autores de la unidad de investigación CECAV recibieron financiamiento de la FCT, en el marco del proyecto UIDB/CVT/0772/2020.Agradecemos a Bruce C. Campbell, Director de Investigación (retirado), Western Regional Research Center, USDA, por su ayuda.

El procedimiento IHQ descrito en este documento es un componente del proyecto de investigación FAIRJ-CT98-7021, "El establecimiento de una red europea para la vigilancia de las EET de rumiantes y la normalización y armonización del proceso y los criterios para la identificación de casos sospechosos". Sigue los criterios diagnósticos definidos por la Organización Mundial de Sanidad Animal, WOAH (anteriormente denominada Office International des Épizooties, OIE)1 y el Laboratorio Europeo de R...

<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>

Las EET son enfermedades zoonóticas potenciales. Tras la aparición de la EEB en 1986 en el Reino Unido, Portugal se convirtió en uno de los Estados miembros de la Unión Europea con mayor incidencia de esta enfermedad14,15. Para controlar esta enfermedad, han surgido otras EET (tembladera clásica y atípica, variantes de la EEB, y actualmente la vigilancia de la desgaste crónica en cérvidos), mecanismos de vigilancia desarrollados por la Dirección General ...

Según la hipótesis del prión, la isoforma anormal (PrPSc) es el componente primario, o único, del agente infeccioso en las encefalopatías espongiformes transmisibles (EET). La confirmación para el diagnóstico de EET se basa en la inmunodetección de PrPSc mediante la aplicación de protocolos de inmunohistoquímica (IHC) y/o métodos de inmunoblot occidentales (WB) de tejidos de encéfalo1.
IHQ es un método que emplea anticuerpos monoclonales o, en algunos casos, policlonales (como anticuerpos primarios) como primer paso en la inmunotinción de antígenos de interés específicamente dirigidos localizados en células de una sección de tejido. Cualquier unión primaria efectiva anticuerpo-antígeno se visualiza utilizando anticuerpos secundarios específicos para los anticuerpos primarios. Estos anticuerpos secundarios se conjugan con enzimas, como la peroxidasa de rábano picante (HRP) o la fosfatasa alcalina (AP). La visualización se logra agregando un sustrato a estas enzimas, produciendo productos de color insolubles localizados en áreas donde los anticuerpos primarios están unidos a los antígenos objetivo. Se puede lograr una mejor visualización mediante la contratinción, en la que se utiliza un tinte para crear un contraste entre el tejido inmunomarcado y el no inmunomarcado2.
Con IHQ utilizando tejidos embebidos en parafina fijados en formalina (FFPE), la fijación de formalina puede anular la efectividad de los anticuerpos primarios debido a la reticulación por formaldehído y el calentamiento y la deshidratación durante la incrustación de parafina. Estos cambian la conformación de las proteínas, destruyendo, desnaturalizando o enmascarando los epítopos, disminuyendo o abrogando así su detección3. Como tal, esto requiere recuperación de antígenos (AR). Las técnicas de AR interrumpen la reticulación del grupo químico relacionado con el formaldehído en las moléculas antigénicas, restaurando o desenmascarando así la conformación antígeno-proteína original. Esto da como resultado la recuperación de la afinidad anticuerpo-antígeno (epítopo) para el inmunomarcaje. La eficacia eventual de la RA depende de las cualidades del antígeno diana y/o del anticuerpo primario2.
La recuperación de antígenos (epítopos) inducidos por calor (HIER) es un procedimiento de AR3 y se usa de forma rutinaria para la detección de IHC de PrPSc , como se describe en este documento. La IHQ es esencial para el diagnóstico y se utiliza en laboratorios de investigación para determinar la distribución tisular de un antígeno asociado a la patología. Es ampliamente utilizado en el diagnóstico e investigación del cáncer, la neurociencia y las enfermedades infecciosas4, entre otros. Para las EET, la IHQ desempeña un papel importante en el diagnóstico y la investigación para confirmar e investigar la distribución de PrPSc en huéspedes naturales y modelos experimentales. IHQ contribuye a los estudios de patogénesis de priones y al análisis de los tipos y patrones de depósito de PrPSc , es decir, en tejidos neurales5, para detectar desviaciones de infecciones descritas rutinariamente e identificar nuevas cepas de priones putativas.
Debido a que los priones de la encefalopatía espongiforme bovina (EEB) pueden infectar a los seres humanos, ciertos protocolos de laboratorio involucrados en el trabajo con la EEB pueden requerir el uso de instalaciones y prácticas BSL-36. Estos incluyen el uso de un recipiente secundario sellado para transportar posibles muestras de tejido infectadas con EEB dentro del instituto y el laboratorio. También incluye la designación de áreas de contención y equipos dedicados a priones para la investigación y el análisis de la EEB siempre que sea posible. Esto se hace para evitar la contaminación fuera del área de trabajo y proporcionar un espacio confinado ya que los procedimientos de descontaminación se hacen necesarios.
En consecuencia, el Laboratorio de Patología del INIAV sigue las instalaciones y prácticas recomendadas de bioseguridad nivel 3 (BSL-3)6 para manejar posibles muestras de tejidos infectados por priones de bovinos, pequeños rumiantes y cérvidos asociados con la vigilancia de EET.
Los tejidos fijados en formalina e incrustados en parafina incluidos en los procedimientos de diagnóstico o investigación de EET, especialmente en el sistema nervioso central, pueden ser potencialmente infecciosos. Por lo tanto, estos tejidos fijos deben tratarse con ácido fórmico para reducir la infectividad de los priones, si están presentes, antes del procesamiento del tejido. Esto se realiza colocando tejidos fijos y recortados (aproximadamente 2-4 mm de espesor) en un casete de procesamiento. El casete se sumerge en ácido fórmico al 98% (durante 1 h). Después de la inmersión, los casetes con pañuelos de papel se lavan en agua corriente del grifo durante 30 minutos y se devuelven al fijador antes de su posterior procesamiento. Si las secciones de tejido no se tratan antes del procesamiento, las secciones de tejido postmicrotómico deben sumergirse en ácido fórmico sin diluir durante al menos 5 minutos antes de la tinción histológica7. El protocolo IHC para PrPSc incluye un paso rutinario de desenmascaramiento de epítopos de ácido fórmico, que también sirve para inactivar los priones7. Después de estos pasos de inactivación de priones, los tejidos fijos resultantes se pueden procesar en BSL-2 utilizando prácticas estándar de BSL-2.
El requisito mínimo de muestreo de tejido para el diagnóstico de EET en cualquier animal incluido en la vigilancia de EET es la recolección del tronco encefálico (a nivel de la ex). Además, para detectar la EEB y la tembladera atípicas, se aconseja que también se recoja parte del cerebelo 1,8. Para el diagnóstico de CWD, tanto los ganglios linfáticos del tronco encefálico (obex) como los retrofaríngeos deben ser probados, ya que PrP Sc podría detectarse en tejidos linfoides sin PrPSc detectable en el obex9, revisado por Machado et al.10.
La porción obex del tronco encefálico incluye sitios diana de diagnóstico de EET, a saber, el núcleo vagal dorsal (DVN), el núcleo del tracto solitario (STN) y el núcleo del tracto espinal del nervio trigémino (V). Estas zonas presentan sistemáticamente acumulación bilateral dePrP Sc , incluso en las primeras fases de la EEB y la tembladera clásica. En los casos clínicos de EET avanzada, todas las áreas de materia gris dentro del tronco encefálico muestran una distribución generalizada de PrPSc 11.
Antes de la sección y el procesamiento, las muestras de cerebro se evalúan (Figura 1) para determinar el nivel de autólisis y la presencia de cualquier daño tisular que pueda comprometer la idoneidad de la muestra para el diagnóstico confirmatorio basado en IHQ8. Para validar la integridad de los protocolos preparatorios y los resultados analíticos, las muestras de tejido positivas y negativas para EET se incluyen como controles junto con la preparación de tejidos a partir de casos de prueba en cada ensayo.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg"> Figura 1: El procedimiento IHC de PrPSc . Representación que muestra la secuencia paso a paso del procedimiento IHC de PrPSc desde la desparafinización de secciones de tejido hasta la eventual inmunotinción y detección (FFPE - Formalin-fixed paraffin-embedded; Mab - anticuerpo monoclonal; DAB - 3,3' diaminobencidina). Esta figura fue creada en BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta 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>
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detection of abnormal prion protein by immunohistochemistry

Las proteínas priónicas anormales (PrPSc) son la isoforma asociada a la enfermedad de la proteína priónica celular y los marcadores diagnósticos de las encefalopatías espongiformes transmisibles (EET). Estas enfermedades neurodegenerativas afectan a los seres humanos y a varias especies animales e incluyen la tembladera, la encefalopatía espongiforme bovina zoonótica (EEB), la desgaste crónica de los cérvidos (CWD) y la recientemente identificada enfermedad priónica del camello (CPD). El diagnóstico de las EET se basa en la inmunodetección de PrPSc mediante la aplicación de métodos de inmunohistoquímica (IHC) e inmunoblot occidental (WB) en los tejidos del encéfalo, es decir, el tronco encefálico (nivel obex). IHQ es un método ampliamente utilizado que utiliza anticuerpos primarios (monoclonales o policlonales) contra antígenos de interés en células de una sección de tejido. La unión anticuerpo-antígeno se puede visualizar mediante una reacción de color que permanece localizada en el área del tejido o célula donde se dirigió el anticuerpo. Como tal, en las enfermedades priónicas, como en otros campos de investigación, las técnicas de inmunohistoquímica no se utilizan únicamente con fines diagnósticos, sino también en estudios de patogénesis. Dichos estudios implican la detección de los patrones y tipos de PrPSc a partir de los descritos anteriormente para identificar las nuevas cepas de priones. Como la EEB puede infectar a los seres humanos, se recomienda que se utilicen instalaciones y/o prácticas de laboratorio de bioseguridad de nivel 3 (BSL-3) para manipular muestras de ganado, pequeños rumiantes y cérvidos incluidas en la vigilancia de las EET. Además, se recomiendan equipos de contención y dedicados a priones, siempre que sea posible, para limitar la contaminación. El procedimiento IHC de PrPSc consiste en un paso de desenmascaramiento de epítopos de ácido fórmico que también actúa como una medida de inactivación priónica, ya que los tejidos fijados en formalina e incrustados en parafina utilizados en esta técnica siguen siendo infecciosos. Al interpretar los resultados, se debe tener cuidado para distinguir el inmunomarcaje no específico del etiquetado objetivo. Para este propósito, es importante reconocer los artefactos de inmunomarcaje obtenidos en animales de control TSE-negativos conocidos para diferenciarlos de los tipos específicos de inmunomarcaje PrPSc , que pueden variar entre cepas de EET, especies hospedadoras y genotipo prnp , descrito más detalladamente en este documento.

Dado que es posible el inmunomarcaje diana no específico, es importante determinar el nivel de inmunomarcaje no específico en animales control con EET negativas conocidas. Este es un paso importante para interpretar adecuadamente el inmunomarcaje específico de PrPSc 7 (Figura 2). Se ha observado que el marcado no diana por anticuerpos anti-PrP ocurre como tinción intraneuronal discreta de partículas finas (más evidente en el núcleo hipoglos...

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

Detection of Abnormal Prion Protein by Immunohistochemistry

El inmunomarcaje de proteínas priónicas anormales mediante protocolos inmunohistoquímicos requiere metodologías específicas de preparación de muestras y anticuerpos anti-PrP. El presente protocolo describe los pasos clave en el desenmascaramiento de epítopos para garantizar un inmunoetiquetado adecuado de PrP y minimizar la tinción de fondo no específica. Además, este enfoque considera medidas de bioseguridad al realizar estudios inmunohistoquímicos con los tejidos infectados por priones.

Detección de proteína priónica anormal por inmunohistoquímica

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

La técnica permite el diagnóstico confirmatorio de enfermedades priónicas mediante la técnica de tembladera PrP en tejidos, analizando los tipos de tembladera PrP y su distribución para evaluar la desviación punitiva al perfil conocido de deposición de tembladera PrP. La técnica está estandarizada y utiliza anticuerpos específicos contra la tembladera PrP, lo que permite la visualización de la deposición de tembladera PrP en los tejidos. La técnica también proporciona información sobre el área neuroanatómica y el tipo de células.Comience colocando los portaobjetos con las secciones de tejido en una canasta de tinción de acero inoxidable. Emerge la cesta en un baño de xileno. Después de tres minutos, retire y sumerja la cesta una vez más.Para eliminar el xileno y rehidratar las secciones, sumerja la cesta en un baño de etanol absoluto. Después de tres minutos, retire y sumerja la cesta una vez más. Seque al aire las secciones antes de colocarlas en un baño de etanol al 90% durante un minuto.A continuación, transfiera la sección a un baño de etanol al 70% durante otro minuto. Para cada tratamiento de baño de etanol, agite suavemente la cesta deslizante dos veces y drene la canasta antes de la próxima transferencia. Transfiera la canasta a un baño de etanol al 50% durante un minuto.Sumerja cuidadosamente las secciones de tejido en ácido fórmico al 98% a temperatura ambiente durante 30 minutos. Enjuague las secciones con agua del grifo durante cinco minutos, seguido de enjuagar dos veces con agua destilada. Use un recipiente de tinción de acero inoxidable en la cámara de presión llena con 500 mililitros de agua destilada para pretratar el tampón de citrato de 10 milimolares a 98 grados centígrados durante 20 minutos.Para fines de control de calidad, coloque una sección de cinta indicadora de autoclave adhesiva en la cesta para controlar la temperatura y la presión. Una vez que la alarma indique que el equipo ha alcanzado el tiempo y la temperatura del programa, sumerja la cesta con portaobjetos. A continuación, inicie el programa en la cámara de presión hasta el punto de ajuste 2 y registre la presión inicial y final de este programa.Para la inactivación de la peroxidasa endógena, sumerja la cesta de portaobjetos que contiene las secciones de muestra en un baño de peróxido de hidrógeno al 3% en metanol durante 30 minutos. Cierre las secciones bajo agua corriente durante cinco minutos Después de drenar, sumerja las secciones en solución salina tamponada con Tris durante cinco minutos adicionales. Para la inmunodetección, coloque cada portaobjetos sobre un soporte de placa de cubierta disponible comercialmente prehumedecido con solución salina tamponada con Tris con el lado del tejido orientado hacia el soporte y los bordes de la diapositiva coincidiendo con los dos puntos inferiores del soporte.Asegúrese de evitar las burbujas de aire. Sostenga el conjunto del portaportaobjetos entre el pulgar y el índice. Mantenga un dedo encima del portaobjetos de muestra y el otro en la parte inferior del soporte, luego coloque el ensamblaje en la galería del sistema.Para asegurarse de que el conjunto esté bien ensamblado, llene el pozo entre el portaobjetos de muestra y el soporte con solución salina tamponada con Tris sin desbordarla. A partir de este punto, asegúrese de que se deben retener aproximadamente 80 microlitros de solución salina tamponada con Tris entre el soporte y el portaobjetos. No permita que las secciones se sequen.Disminuya la tinción de fondo antes del tratamiento con el anticuerpo primario incubando previamente las muestras de portaobjetos durante 30 minutos con suero normal al 20% de la misma especie que el huésped de anticuerpos secundario en solución salina tamponada con Tris. Sin lavar las secciones, aplique 200 microlitros de la solución de anticuerpos primarios directamente en cada pocillo del juego portaportaobjetos e incube durante 60 minutos a temperatura ambiente. Para lavar las secciones, llene los pocillos con solución salina tamponada con Tris y espere cinco minutos.Repita el lavado dos veces. A continuación, diluya el anticuerpo secundario biotinilado a una concentración de 1 a 200 en solución salina tamponada con Tris con suero de caballo al 10%. Reparar el volumen requerido en función del número de secciones a tratar.Aplique 200 microlitros de solución de anticuerpos secundarios en cada pocillo del juego portaportaobjetos. Después de 30 minutos de incubación a temperatura ambiente, repita el lavado salino tamponado con Tris. Para la incubación con peroxidasa del complejo avidina-biotina, prepare el reactivo 30 minutos antes de su uso y aplique 200 microlitros de la solución en cada pocillo del soporte de la placa portaobjetos.Una vez hecho esto, incubar el soporte de la placa durante 30 minutos a temperatura ambiente. Se determinó el nivel de inmunomarcaje no específico en animales control con encefalopatías espongiformes transmisibles conocidas negativas para interpretar adecuadamente el inmunomarcaje específico de proteínas priónicas anormales. Se observó que el marcado no objetivo por anticuerpos anti-PrP se producía como tinción intraneuronal discreta de partículas finas, hilos lineales finos irregulares de tinción en el neuropilo, marcado difuso no particulado en algunas áreas de materia gris y blanca, y marcado citoplasmático difuso de neuronas.La tabla resume la descripción de los tipos de proteínas priónicas anormales inmunomarcadas observadas en la encefalopatía espongiforme bovina, la tembladera clásica y atípica, y una enfermedad de desgaste crónico de los cérvidos para un diagnóstico positivo y criterios establecidos para resultados negativos, no concluyentes e inadecuados. Todos los pasos son muy importantes. El pH de las soluciones y la temperatura ambiente son características importantes para el éxito de esta técnica.El desarrollo de inmunohistoquímica para detectar tanto la tembladera de PrP como el tipo de células puede proporcionar información sobre cada célula involucrada en la patogénesis principal.

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 combinación de colorantes lipofílicos, In situ</emHibridación>, inmunohistoquímica y la histología

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

Investigación

Neurociencias

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.

La detección de la hipoxia Microrregional en corteza cerebral de ratón por la imagen de dos fotones de fluorescencia endógena 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.

La inmunohistoquímica wholemount de Topografía Revelando Complex Brain

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.

La disección e inmunohistoquímica de larva, pupa y adulto<em&gt; Drosophila</em&gt; Retinas

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.

La detección de palmitoilación proteínas en neuronas del hipocampo cultivadas por inmunoprecipitación e Intercambio Acyl-Biotina (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.

Detección de<em&gt; In Situ</em&gt; Complejos proteína-proteína en la<em&gt; Drosophila</em&gt; Larval unión neuromuscular Usando proximidad Ligadura Ensayo

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.

Imágenes de bioluminiscencia de la neuroinflamación en ratones transgénicos Después periférica La inoculación de la alfa-sinucleína fibrillas

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.

Monitoreo de la transmisión de célula a célula de los agregados de la proteína Prion-como en 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.

Cuantificar la morfología de la microglía de microfotografías de la inmunohistoquímica preparado tejido con 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.

Método autoradiográfico cuantitativo para la determinación de las tasas regionales de síntesis de proteínas cerebrales 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 ...