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

이 기사는 FCT(Fundação para a Ciência e a Tecnologia) - FEDER -Balcão2020이 지원하는 프로젝트 POCI-01-0145-FEDER-029947 "포르투갈의 만성 소모성 질병 위험 평가"의 자금 지원을 받았습니다. 또한 연구 단위 CECAV의 저자는 UIDB/CVT/0772/2020 프로젝트에 따라 FCT로부터 자금을 받았습니다.

여기에 설명 된 IHC 절차는 연구 프로젝트 FAIRJ-CT98-7021, "반추 동물 TSE의 감시를위한 유럽 네트워크 구축 및 의심 사례 식별을위한 프로세스 및 기준의 표준화 및 조화"의 구성 요소입니다. 이는 세계동물보건기구(World Organization for Animal Health), WOAH(이전에는 Office International des Épizooties, OIE)1 및 동물 TSE에 대한 유럽 참조 실험실(European Reference Laboratory for animal TSEs 8,11<sup class="...

<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>

TSE는 잠재적인 인수공통전염병입니다. 영국에서 1986에서 BSE가 출현 한 후, 포르투갈은이 질병의 발병률이 높은 유럽 연합 회원국 중 하나가되었습니다14,15. 이 질병을 통제하기 위해 등장한 다른 TSE (고전적 및 비정형 스크래피, BSE 변종 및 현재 자궁 경부에서 만성 소모성 질환의 감시), 감시 메커니즘은 식품 수의학 총국 (DGAV)과 국립 농업 및 수의학 연...

프리온 가설에 따르면, 비정상적인 동형(PrPSc)은 전염성 해면상 뇌병증(TSE)에서 감염원의 주요 또는 유일한 구성 요소입니다. TSE 진단을 위한 확인은 면역조직화학(IHC) 프로토콜 및/또는 뇌 조직의 웨스턴 면역블롯 방법(WB)을 적용하여 PrPSc 의 면역검출에 의존합니다1.
IHC는 조직 절편의 세포에 위치하는 특이적 표적화된 관심 항원의 면역염색의 첫 번째 단계로서 단일클론 항체 또는 일부 경우에 다클론 항체(1차 항체)를 사용하는 방법입니다. 임의의 효과적인 1차 항체-항원 결합은 1차 항체에 특이적인 2차 항체를 사용하여 시각화된다. 이러한 2차 항체는 양 고추 냉이 과산화 효소 (HRP) 또는 알칼리성 포스파타제 (AP)와 같은 효소에 접합됩니다. 그런 다음 이러한 효소에 기질을 추가하여 1차 항체가 표적 항원에 결합하는 영역에 국한된 불용성 색상 제품을 생성함으로써 시각화를 달성합니다. 개선된 시각화는 대조염색에 의해 달성될 수 있으며, 여기서 염료는 면역표지된 조직과 면역표지되지 않은 조직 사이의 대조를 생성하기 위해 사용된다2.
포르말린 고정 파라핀 포매 조직 (FFPE)을 사용하는 IHC를 사용하면 포르말린 고정은 포름알데히드에 의한 가교 결합 및 파라핀 포매 동안 가열 및 탈수로 인한 1차 항체의 효과를 무효화할 수 있습니다. 이들은 단백질의 형태를 변화시켜 에피토프를 파괴, 변성 또는 마스킹하여 검출을 감소시키거나 폐지한다3. 따라서 항원 검색(AR)이 필요합니다. AR 기술은 항원 분자에서 포름알데히드 관련 화학 그룹 가교를 방해하여 원래의 항원-단백질 형태를 복원하거나 마스킹을 해제합니다. 이는 면역표지를 위한 항체-항원(에피토프) 친화도를 회복시키는 결과를 가져온다. AR의 최종 효능은 표적 항원 및/또는 1차 항체의 특성에 따라 달라집니다2.
열-유도 항원 (에피토프) 검색 (HIER)은 AR3 의 한 절차이고, 본원에 기재된 바와 같이,PrPSc IHC 검출을 위해 일상적으로 사용된다. IHC는 진단에 필수적이며 병리학 관련 항원의 조직 분포를 결정하기 위해 연구 실험실에서 사용됩니다. 암, 신경과학, 전염병4 등의 진단 및 연구에 널리 사용된다. TSE의 경우, IHC는 자연 숙주 및 실험 모델에서 PrPSSc 분포를 확인하고 조사하기 위한 진단 및 연구에서 중요한 역할을 합니다. IHC는 프리온 병인 연구와PrPSC 침착 유형 및 패턴, 즉 신경 조직5의 분석에 기여하여 일상적으로 기술되는 감염으로부터의 편차를 감지하고 추정되는 새로운 프리온 균주를 식별한다.
소 해면상 뇌병증(bovine spongiform encephalopathy, BSE)의 프리온은 인간을 감염시킬 수 있기 때문에, BSE 연구와 관련된 특정 실험실 프로토콜은 BSL-3 시설 및 관행을 사용해야 할 수 있다6. 여기에는 밀봉된 2차 용기를 사용하여 연구소 및 실험실 내에서 잠재적인 BSE 감염 조직 샘플을 운반하는 것이 포함됩니다. 또한 가능할 때마다 BSE 연구 및 분석을 위한 격리 구역과 프리온 전용 장비를 지정하는 것도 포함됩니다. 이는 작업장 외부의 오염을 방지하고 오염 제거 절차가 필요하기 때문에 밀폐된 공간을 제공하기 위해 수행됩니다.
따라서, INIAV의 병리학 실험실은 TSE 감시와 관련된 소, 작은 반추 동물 및 자궁 경부의 잠재적인 프리온 감염 조직 샘플을 관리하기 위해 권장되는 생물안전 등급-3(BSL-3) 시설 및 관행6 을 따른다.
TSE 진단 또는 연구 절차, 특히 중추 신경계에 포함 된 포르말린 고정 및 파라핀 포매 조직은 잠재적으로 전염 될 수 있습니다. 따라서 이러한 고정 조직은 조직 처리 전에 프리온의 감염성을 줄이기 위해 포름산으로 처리해야 합니다. 이것은 고정되고 손질 된 조직 (약 2-4mm 두께)을 처리 카세트에 넣어 수행됩니다. 그런 다음 카세트를 98% 포름산에 담급니다(1시간 동안). 침지 후, 티슈가 있는 카세트를 흐르는 수돗물에서 30분 동안 세척하고, 추가 처리 전에 고정액으로 되돌립니다. 조직 절편을 처리 전에 처리하지 않는 경우, 조직학적 염색 전에 최소 5분 동안 희석되지 않은 포름산에 세포 절편을 담가야 한다7. PrPSc 에 대한 IHC 프로토콜은 일상적인 포름산 에피토프-데마스킹 단계를 포함하며, 또한 프리온을 불활성화시키는 역할을 한다(7). 이러한 프리온 불활성화 단계 후, 생성된 고정 조직은 표준 BSL-2 관행을 사용하여 BSL-2에서 처리될 수 있습니다.
TSE 감시에 포함 된 모든 동물에서 TSE 진단을위한 최소 조직 샘플링 요구 사항은 뇌간을 수집하는 것입니다 (obex 수준에서). 또한, 비정형 BSE와 스크래피를 검출하기 위해서는 소뇌의 일부도 채취하는 것이 좋다 1,8. CWD 진단의 경우, Machado et al.10에 의해 검토된 obex9에서 검출 가능한 PrP Sc가 없는 림프 조직에서 PrPSc가 검출될 수 있으므로 뇌간(obex)과 후인두 림프절을 모두 검사해야 합니다.
뇌간의 obex 부분에는 진단 TSE 표적 부위, 즉 삼차 신경의 등쪽 미주 신경(DVN), 고립관 핵(STN) 및 척수 핵이 포함됩니다. 이 영역은 BSE 및 고전적 스크래피의 초기 단계에서도 일관되게 양측 PrPSc 축적을 나타냅니다. 진행성 TSE의 임상 사례에서, 뇌간 내의 모든 회백질 영역은 광범위한 PrPS분포 를 보인다11.
절편 및 처리 전에, 뇌 검체를 평가하여(그림 1) 자가분해 수준과 IHC 기반 확증 진단에 대한 검체의 적합성을 잠재적으로 손상시킬 수 있는 조직 손상의 존재를 확인한다8. 분취 프로토콜 및 분석 결과의 무결성을 검증하기 위해 TSE 양성 및 음성 조직 샘플은 각 분석에서 테스트 케이스의 조직 준비와 함께 대조군으로 포함됩니다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg"> 그림 1: PrPSc IHC 절차. 조직 절편의 탈파라핀화부터 최종 면역염색 및 검출까지PrPSC 의 단계별 서열을 보여주는 IHC 절차(FFPE - Formalin-fixed paraffin-embedded; Mab - 단클론 항체; DAB - 3,3' 디아미노벤지딘). 이 그림은 BioRender.com 년에 만들어졌습니다. <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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

비정상 프리온 단백질(PrP,Sc)은 세포 프리온 단백질의 질병 관련 동형이며 전염성 해면상 뇌병증(TSE)의 진단 마커입니다. 이러한 신경퇴행성 질환은 인간과 여러 동물 종에 영향을 미치며 스크래피, 인수공통전염병(BSE), 만성 자궁경부 소모성 질환(CWD) 및 새로 확인된 낙타 프리온 질병(CPD)을 포함합니다. TSE의 진단은 뇌 조직, 즉 뇌간(obex 수준)에 면역조직화학(IHC)과 웨스턴 면역블롯 방법(WB)을 모두 적용하여 PrPSc의 면역검출에 의존합니다. IHC는 조직 절편의 세포에서 관심있는 항원에 대해 1 차 항체 (단일 클론 또는 폴리 클로날)를 사용하는 널리 사용되는 방법입니다. 항체-항원 결합은 항체가 표적이 된 조직 또는 세포 영역에 국한된 채로 남아 있는 색 반응에 의해 시각화될 수 있습니다. 따라서 프리온 질병에서 다른 연구 분야와 마찬가지로 면역 조직 화학 기술은 진단 목적뿐만 아니라 병인 연구에도 사용됩니다. 이러한 연구는 새로운 프리온 균주를 식별하기 위해 이전에 설명한 것에서 PrPSc 패턴 및 유형을 검출하는 것을 포함합니다. BSE는 인간을 감염시킬 수 있으므로 TSE 감시에 포함된 소, 작은 반추 동물 및 자궁경부 샘플을 처리하기 위해 생물안전 실험실 레벨 3(BSL-3) 시설 및/또는 관행을 사용하는 것이 좋습니다. 또한 오염을 제한하기 위해 가능하면 봉쇄 및 프리온 전용 장비를 권장합니다. PrPSc IHC 절차는 이 기술에 사용된 포르말린 고정 및 파라핀 포매 조직이 감염성을 유지하기 때문에 프리온 불활성화 측정으로도 작용하는 포름산 에피토프 데마스킹 단계로 구성됩니다. 결과를 해석할 때 비특이적 면역 표지와 표적 표지를 구별하기 위해 주의를 기울여야 합니다. 이러한 목적을 위해, 본원에 추가로 기술된 TSE 균주, 숙주 종 및 prnp 유전자형 간에 변할 수 있는 특정 PrPSSc 면역표지 유형으로부터 이들을 구별하기 위해 공지된 TSE-음성 대조군 동물에서 얻어진 면역표지의 인공물을 인식하는 것이 중요하다.

비특이적 표적 면역 표지가 가능하다는 점을 감안할 때, 알려진 TSE 음성 대조군 동물에서 비특이적 면역 표지의 수준을 확인하는 것이 중요합니다. 이는 특정PrPSc 면역표지를 적절하게 해석하기 위한 중요한 단계이다7(그림 2). 항-PrP 항체에 의한 비표적 표지는 별개의 신경내 미세 미립자 염색(설하 핵 및 부속 교미 핵에서 더 분명함), 신경?...

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

Detection of Abnormal Prion Protein by Immunohistochemistry

면역조직화학 프로토콜을 사용하여 비정상 프리온 단백질을 면역표지하려면 특정 샘플 및 항-PrP 항체 준비 방법론이 필요합니다. 본 프로토콜은 적절한 PrP 면역표지를 보장하고 비특이적 배경 염색을 최소화하기 위한 에피토프 마스킹의 주요 단계를 설명합니다. 또한, 이 접근법은 프리온 감염 조직으로 면역조직화학 연구를 수행할 때 생물학적 안전성 조치를 고려합니다.

면역조직화학에 의한 비정상적인 프리온 단백질 검출

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

이 기술은 조직에서 PrP 스크래피 기술에 의해 프리온 질병의 확증 진단을 가능하게 하고, PrP 스크래피 유형 및 분포를 분석하여 알려진 PrP 스크래피 침착 프로파일에 대한 징벌적 편차를 평가할 수 있습니다. 이 기술은 표준화되어 있으며 PrP 스크래피에 대한 특정 항체를 사용하여 조직에서 PrP 스크래피 증착을 시각화할 수 있습니다. 이 기술은 또한 신경 해부학 적 영역과 세포 유형에 대한 정보를 제공합니다.조직 섹션이 있는 슬라이드를 스테인리스 스틸 염색 바구니에 넣는 것으로 시작합니다. 크실렌 욕조에서 바구니를 꺼냅니다. 3 분 후 바구니를 꺼내 다시 한 번 담그십시오.자일렌을 제거하고 섹션을 재수화하려면 바구니를 절대 에탄올 욕조에 담그십시오. 3 분 후 바구니를 꺼내 다시 한 번 담그십시오. 섹션을 90% 에탄올 욕조에 1분 동안 넣기 전에 자연 건조시킵니다.다음으로, 섹션을 70 % 에탄올 욕조로 1 분 더 옮깁니다. 각 에탄올 수조 처리에 대해 슬라이드 바스켓을 부드럽게 두 번 저어주고 다음 이송 전에 바스켓을 비우십시오. 바구니를 50% 에탄올 수조에 1분 동안 옮깁니다.조직 절편을 실온에서 30분 동안 98% 포름산에 조심스럽게 담그십시오. 섹션을 수돗물로 5분 동안 헹구고 증류수로 두 번 헹굽니다. 500 밀리리터의 증류수로 채워진 압력 챔버에서 스테인레스 스틸 염색 용기를 사용하여 섭씨 98도에서 20 분 동안 10 밀리몰 구연산염 완충액을 전처리한다.품질 관리를 위해 접착제 오토클레이브 표시기 테이프 부분을 바스켓에 올려 온도와 압력을 모니터링합니다. 알람이 장비가 프로그램 시간과 온도에 도달했음을 나타내면 슬라이드로 바구니를 담그십시오. 그런 다음 압력 챔버에서 설정 포인트 2까지 프로그램을 시작하고 이 프로그램의 초기 및 최종 압력을 기록합니다.내인성 과산화효소의 비활성화를 위해 샘플 섹션이 들어 있는 슬라이드 바스켓을 메탄올의 3% 과산화수소 수조에 30분 동안 담그십시오. 흐르는 물에 5분 동안 다리를 놓으십시오. 배수 후 절편을 트리스 완충 식염수에 추가로 5분 동안 담그십시오. 면역 검출을 위해 조직 측면이 홀더를 향하고 슬라이드 가장자리가 홀더의 두 하단 지점과 일치하도록 트리스 완충 식염수로 미리 적신 시중에서 판매되는 커버 플레이트 홀더에 각 슬라이드를 놓습니다.기포를 피하십시오. 엄지와 집게손가락 사이에 슬라이드 홀더 어셈블리를 잡습니다. 한 손가락을 샘플 슬라이드 위에 놓고 다른 손가락을 홀더 하단에 놓은 다음 어셈블리를 시스템 갤러리에 놓습니다.세트가 잘 조립되었는지 확인하려면 샘플 슬라이드와 홀더 사이의 웰을 트리스 완충 식염수로 넘치지 않게 채웁니다. 이 시점부터 홀더와 슬라이드 사이에 약 80마이크로리터의 트리스 완충 식염수가 유지되어야 합니다. 섹션이 건조되지 않도록 하십시오.1차 항체로 처리하기 전에 슬라이드 샘플을 트리스 완충 식염수에서 2차 항체 숙주와 동일한 종의 20% 정상 혈청으로 30분 동안 사전 배양하여 배경 염색을 감소시킵니다. 절편을 세척하지 않고, 200 마이크로리터의 1차 항체 용액을 슬라이드-홀더 세트의 각 웰에 직접 적용하고, 실온에서 60분 동안 인큐베이션한다. 섹션을 씻으려면 트리스 완충 식염수로 우물을 채우고 5 분 동안 기다리십시오.세탁을 두 번 반복하십시오. 다음으로, 비오티닐화된 2차 항체를 10% 말 혈청으로 트리스 완충 식염수에 1-200 농도로 희석합니다. 치료할 섹션의 수에 따라 필요한 볼륨을 수리하십시오.200 마이크로리터의 2차 항체 용액을 슬라이드 홀더 세트의 각 웰에 적용한다. 실온에서 30분 동안 배양한 후 트리스 완충 식염수 세척을 반복합니다. 아비딘-비오틴 복합체 과산화효소로 배양하기 위해 사용 30분 전에 시약을 준비하고 슬라이드 플레이트 홀더의 각 웰에 200마이크로리터의 용액을 도포합니다.완료되면 플레이트 홀더 세트를 실온에서 30분 동안 배양합니다. 알려진 전염성 해면상 뇌병증-음성 대조군 동물에서 비특이적 면역표지의 수준은 특이적 비정상 프리온 단백질 면역표지를 적절하게 해석하기 위해 결정되었습니다. 항-PrP 항체에 의한 비표적 표지는 개별적인 뉴런 내 미세 미립자 염색, 뉴로필의 불규칙한 미세 선형 염색, 일부 회백질 영역에서의 확산 비미립자 라벨링, 뉴런의 확산 세포질 라벨링으로 발생하는 것으로 나타났습니다.이 표는 양성 진단을 위한 소 해면상 뇌병증, 고전적 및 비정형 스크래피, 자궁경부의 만성 소모성 질환에서 관찰되는 면역 표지된 비정상 프리온 단백질 유형에 대한 설명과 음성, 결정적이지 않고 부적절한 결과에 대한 확립된 기준을 요약합니다. 모든 단계는 매우 중요합니다. 용액의 pH와 실온은 이 기술의 성공을 위한 중요한 특징입니다.PrP 스크래피와 세포 유형을 모두 검출하기 위한 면역조직화학을 개발하면 주요 발병기전에 관여하는 각 세포에 대한 정보를 얻을 수 있습니다.

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.

Lipophilic 염료를 결합, 현장에서 하이브리드화, Immunohistochemistry, 그리고 조직학

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

연구

신경과학

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.

내생 NADH 형광의 2 광자 이미징에 의한 마우스 대뇌 피질의 Microregional Hypoxia의 탐지

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.

의미 심장한 복잡한 두뇌 지형에 대한 Wholemount Immunohistochemistry

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.

애벌레의, Pupal 및 성인의 해부 및 Immunohistochemistry<em&gt; Drosophila</em&gt; 망막

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.

Immunoprecipitation 및 아실-비오틴 거래소 (아베)에 의한 양식 Hippocampal 뉴런의 단백질 Palmitoylation의 감지

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.

검출<em&gt; 현장에서</em상기&gt; 단백질 - 단백질 복합체<em&gt; 초파리</em근접 내고 분석을 사용하기&gt; 애벌레 신경 근육 접합부

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.

알파 - 시누 클레인 브릴의 주변 접종 후 형질 전환 마우스의 신경 염증의 생물 발광 영상

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.

초파리 Melanogaster 에서 프리온과 같은 단백질의 셀 전송 모니터링

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.

Microglia 형태학 Immunohistochemistry 조직 ImageJ를 사용 하 여 준비의 Photomicrographs에서 측정

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.

생체 내 뇌 단백질 합성의 지역 비율 측정을 위한 정량적 자가 방사선 측정 방법

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