We thank Hans Lassmann and Jan Bauer for their guidance and support. We also thank Katalin Benedek for excellent technical assistance and Caroline Westerlund for critical and linguistic appraisal.
This study was supported by grants from Biogen Idec, the Wenner-Gren Foundation, the Swedish Research Council, the Swedish Association of Persons with Neurological Disabilities, Swedish Brain Foundation, the EU 6TH Framework EURATools (LSHG-CT-2005-019015) and Neuropromise (LSHM-CT-2005-018637). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

윤리 정책 본 연구는 실험 동물에 대한 스웨덴 국립 이사회와 유럽 공동체위원회 지침의 지침에 따라 수행되는 윤리적 허가에 따라 (6백9분의 86 / EEC)는 N338 / 09, N15 / 10 N65 / 10에 의해 승인 된 북한 스톡홀름 동물 윤리위원회. 1. 조직 준비 <ol><li> 관류 및 고정 <ol><li> 좌심실을 통하여 관류 transcardial 수행 이소 플루 란 동물을 마취. 0.1 M PBS (PFA)에 4 % 파라 포름 알데히드 다음 혈액 성분을 제거하기 위해 인산 완충 식염수 (PBS)와 혈관의 헹굼을 시작합니다. </li><li> PFA에 침지하여 해부 뇌, 척수 및 말초 LN 조직을 흥분시키는. 4 ° C에서 24 시간 후, 추가 처리 할 때까지 4 ° C에서 PBS에 PFA에서 조직과 상점을 전송합니다. 또는 상업적 PBS에, 250 ml의의 S &amp;에 9g의 NaCl을 용해# 246; rensen은 버퍼 750 ml의를 탈 이온수를 추가 (DH 2 O); 2.5 L의 DH 2 O.에서 HPO 2 PO 4 X 2H 2 O를 4 × 상반기 2 O 및 71.2 g 2 나 0.2 M 소렌슨 완충액 (pH 7.4) 용해 13.8 g의 NaH를 준비합니다 </li></ol></li><li> 탈수 및 삽입 <ol><li> 면도날을 사용하여 약 5 ㎜의 두께 부분으로 조직을 잘라. </li><li> 크실렌 최종적 파라핀 (표 1) 다음에 조직 테크 VIP 진공 침투 프로세서 (에탄올의 농도 상승에 침수에 기초한 표준 절차를 사용하여 티슈 경화 처리 (탈수)을 시작합니다. </li><li> 금형에 조직을 올려 놓고 &quot;파라핀 블록&quot;을 형성하기 위해 샘플 주위 유동 파라핀에 붓는다. 장기 보관은 실온 (RT)가 필요합니다. 그러나, 절편하기 전에, 하룻밤, 예를 들어, 4 ℃에서 (O / N) 블록을 냉각하기를 권장합니다.</li></ol></li></ol> 2. 단면 <ol><li> 칼에 걸쳐 앞뒤로 이동할 수 썰매 마이크로톰의 고정 홀더로 파라핀 블록을 배치합니다. 블록 및 마이크로톰 나이프 (즉, 칼의 형상에 따라, 또한 절단 속도 및 기법) 사이에 최적의 각도를 조정한다. </li><li> 파라핀 블록에서 5 μm의 두께 단면 - 3 컷. </li><li> 물 용기에 바로 절단 부분을 전송하고 유리 슬라이드에 물에서 연속적으로 마운트. </li><li> 잔류 물과 잠재적 인 기포를 제거하기 위해 종이 타월에 대해 조심스럽게 장착 섹션을 누릅니다. 상업적으로 미리 접착 코팅 된 유리 슬라이드를 추천한다. </li><li> 60 °의 C - 드라이 (50)에 난로에서 두 시간 동안 슬라이드를 장착. </li></ol> 3. 탈 파라핀 (재수 및 차단 내생 퍼 옥시 다제) <ol><li> 슬라이드는 자일 렌으로 2 배 빠져 (또는 자일 렌 대체XEM-200), 각 시간 15-20 &#39;. </li><li> 99 % 에탄올로 씻어. </li><li> 내인성 퍼 옥시 다제 활성을 차단하기 위해, 0.25 %의 과산화수소를 함유하는 메탄올 용액 30 &#39;에 대한 섹션을 배양한다. </li><li> 수분 함량 (99 %, 70 % 증가하고, 증류수에서 끝나는 에탄올을 사용하여 재수 화를 계속한다. </li><li> 커버 장착 될 때까지이 시점에서 섹션을 촉촉하게 유지해야 할 전표. </li></ol> 4. 항원 검색 <ol><li> 작업을 준비하는 단계; 60 &#39;(이 경우에는 EDTA의 pH 8.5 완충액) 항원 검색 용액 슬라이드 비등하는 찜통을 사용 (EDTA 완충액 원액 DH 2 O 50 ㎖에 용해시킨 1.21 g 트리스 및 0.37 g의 EDTA를 포함 47.5 ml의 DH 2 O) 용액의 희석 2.5ml를의 EDTA 원액. </li><li> 약에 대한 RT에 슬라이드를 냉각. 1 시간 후, 3 린스 - 트리스와 배는 생리 식염수 (TBS, 사용 대안 PBS)를 0.05 M 트리스 0.15 M의 NaCl로 구성된 완충; pH를 7.5으로 조정 위스콘신일 염산. 또는 상업적 TBS를 사용합니다. </li></ol> 5. 비특이적 결합 부위를 차단 <ol><li> , 비특이적 백그라운드 반응을 피하기 10 % 소 태아 혈청 (FCS) 및 90 % DAKO 완충액을 함유하는 차단 용액에 &#39;30 RT에서 섹션을 배양. </li></ol> 6. 두 번 Immunolabeling : 기본 애비와 동시 배양 <ol><li> 차단 솔루션 차 항체의 필요한 양을 희석 및 O / n를 4 ° C에서 품어. (1 ED1 : 1000) α-Cd68 또는 α-Iba1 (Aif1, 1 : 1000) 또는 α-Cd8α (황소 8; 1 : 200); 이중 면역 염색에 대한 α-에오 탁신 (300 일 Ccl11)를 ​​결합 . </li><li> 슬라이드는 TBS 완충액으로 3 ~ 5 배 린스 (또는 10 배 희석 DAKO 버퍼를 사용한다). </li><li> 차단 솔루션과 RT에 1 시간을 품어 : 이차 항체의 필요량 (200 바이오틴 안티 염소와 AP-복합 항 - 마우스, 모두 1) 희석. </li><li> (우리를 TBS 버퍼와 5 배 - 슬라이드 3을 씻어대안 예 10 배) DAKO 버퍼를 희석시켰다. </li><li> RT에서 1 시간 동안 블로킹 용액으로 희석하고 아비딘 - 고추 냉이 퍼 옥시 다제 착체 (HRP)와 함께 인큐베이션 슬라이드. </li><li> 슬라이드 3 린스 - TBS 버퍼 (또는 10 배 희석 DAKO 버퍼를 사용)으로 5 배. </li></ol> 7. 시각화 <ol><li> 바운드 AP 표지 이차 항체의 시각화 <ol><li> 1 L의 DH 2 O 12.1 g의 트리스 용해시켜 0.1 M 트리스 HCl 완충액을 준비하고 염산을 이용하여 pH를 조절한다. 레바 미솔 1 M 용액을 제조하기 위해 동일한 트리스 -HCl 버퍼를 사용한다. DH 2 O.에서 갓 4 %에 NaNO 2 용액을 제조 </li><li> (37 (하나의 표준 유리 큐벳에 필요한 볼륨) 빠른 블루 (FB) 기판 유리 튜브 312.5 μL의 DMF에 6.25 mg의 나프톨-AS-MX-인산을 용해 및 사전 예열 50 ㎖로 저어 50 ㎖를 얻으려면 ° C) 트리스 - 염산 완충액. 312.5 ㎕의 2 N 염산 12.5 mg의 FB RR 소금을 용해하는 것은 처리장 이전의 312.5 μl를 추가4 %에 NaNO 2 용액은 적색 및 예열 트리스 - 염산 완충액 동일한 50 ㎖로 혼합 교반한다. 노란 액체가 분명해질 때까지 가볍게 흔들어 마지막으로 이전에 제조 된 1 M의 levamisole 용액 77 μl를 추가합니다. 여액의 혼합물을 수득하고, 유리 큐벳에 넣고 슬라이드 위에 붓는다. </li><li> 37 ° C에서 배양을 시작하고 약 광학 현미경 하에서 공정을 개발하고 제어 할 수 있습니다. 매 15-30 분. 퍼지 온 경우, 새로운 혼합물로 FB 솔루션을 교체합니다. </li><li> PBS 버퍼에이어서 TBS 버퍼 및 전송에 5 배 - 슬라이드 3을 씻어. </li></ol></li><li> 결합 된 바이오틴 이차 항체의 시각화 <ol><li> DAB는 / H 2 O 2 49 ml의 PBS 1 ml의 DAB 원액 (1 ml의 PBS 당 25 mg을 DAB)을 희석하여 솔루션을 개발 준비합니다. 쏟아져 이전 섹션에 16.5 μL의 H 2 O 2 및 여과 액을 추가합니다. </li><li> 침전 이마에 발색의 DAB의 전환n 개의 안료는 즉시 할 수 있습니다. (높은 배경을 높이고 특정 신호를 마스크 수 초 이상 배양에도 몇) 광 현미경 개발 프로세스를 제어 할 수 있습니다. </li><li> 갈색 안료 석출물의 강도는 대안 적으로 &#39;5 2 % 황산구리 및 0.9 % 염화나트륨 용액으로 이루어진 배양을 향상시킬 수있다. </li><li> 슬라이드 3 린스 - PBS로 5 배 그리고 마지막으로 DH 2 O와 함께, 또는 수돗물을 사용합니다. </li><li> 커버 수성 GelTol 설치 매체를 사용하여 물에서 직접 전표와 슬라이드를 탑재합니다. 공기 방울을 생성하지 마십시오. </li><li> 설치 매체의 완전한 건조, 예를 들어, O / N에서 4 ℃로 할 수 있습니다. 스토어 RT에 슬라이드를 건조. </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1. </td><td> % 에탄올 </td><td> 20 &#39; </td><td> 40 ° C </td></tr><tr><td> 2. </td><td> % 에탄올 </td><td> 60# 39; </td><td> 40 ° C </td></tr><tr><td> 삼. </td><td> % 에탄올 </td><td> 90 &#39; </td><td> 40 ° C </td></tr><tr><td> 4. </td><td> % 에탄올 </td><td> 60 &#39; </td><td> 40 ° C </td></tr><tr><td> 5. </td><td> % 에탄올 </td><td> 90 &#39; </td><td> 40 ° C </td></tr><tr><td> 6. </td><td> % 에탄올 </td><td> 60 &#39; </td><td> 40 ° C </td></tr><tr><td> 7. </td><td> % 에탄올 </td><td> 90 &#39; </td><td> 40 ° C </td></tr><tr><td> 8. </td><td> % 에탄올 </td><td> 120 &#39; </td><td> 40 ° C </td></tr><tr><td> 9. </td><td> 크 실롤 </td><td> 30 &#39; </td><td> 40 ° C </td></tr><tr><td> 10. </td><td> 크 실롤 </td><td> 60 &#39; </td><td> 40 ° C </td></tr><tr><td> 11. </td><td> 파라핀 </td><td> 60 &#39; </td><td> 60 ° C </td></tr><tr><td> 12. </td><td> 파라핀 </td><td> 60 &#39; </td><td> 60 ° C </td></tr><tr><td> 1삼. </td><td> 파라핀 </td><td> 60 &#39; </td><td> 60 ° C </td></tr><tr><td> 14. </td><td> 파라핀 </td><td> 120 &#39; </td><td> 60 ° C </td></tr></tbody></table> 조직 테크 VIP 진공 침투 프로세서에 의해 표 1. 조직 처리.

<ol>
	<li>Coons, A., Creech, H. J., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+properties+of+an+antibody+containing+a+fluorescent+group.">Immunological properties of an antibody containing a fluorescent group.</a> Proc Soc Exp Biol Med. 47, 200-202 (1941).</li><li>Nakane, P. K., Pierce, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-labeled+antibodies:+preparation+and+application+for+the+localization+of+antigens.">Enzyme-labeled antibodies: preparation and application for the localization of antigens.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 14, 929-931 (1966).</li><li>Avrameas, S., Uriel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+antigen+and+antibody+labelling+with+enzymes+and+its+immunodiffusion+application.">Method of antigen and antibody labelling with enzymes and its immunodiffusion application.</a> C R Acad Sci Hebd Seances Acad Sci D. 262, 2543-2545 (1966).</li><li>Mason, D. Y., Sammons, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+preparation+of+peroxidase:+anti-peroxidase+complexes+for+immunocytochemical+use.">Rapid preparation of peroxidase: anti-peroxidase complexes for immunocytochemical use.</a> Journal of immunological. 20, 317-324 (1978).</li><li>Faulk, W. P., Taylor, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunocolloid+method+for+the+electron+microscope.">An immunocolloid method for the electron microscope.</a> Immunochemistry. 8, 1081-1083 (1971).</li><li>Coons, A. H., Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+antigen+in+tissue+cells;+improvements+in+a+method+for+the+detection+of+antigen+by+means+of+fluorescent+antibody.">Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody.</a> The Journal of experimental medicine. 91, 1-13 (1950).</li><li>Polak, J. M., Van Noorden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to immunocytochemistry. , (2003).</li><li>Elias, J. M., Margiotta, M., Gaborc, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+and+detection+efficiency+of+the+peroxidase+antiperoxidase+(PAP),+avidin-biotin+peroxidase+complex+(ABC),+and+peroxidase-labeled+avidin-biotin+(LAB)+methods.">Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin peroxidase complex (ABC), and peroxidase-labeled avidin-biotin (LAB) methods.</a> American journal of clinical pathology. 92, 62-67 (1989).</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=Technical+aspects+of+immunohistochemistry.">Technical aspects of immunohistochemistry.</a> Vet Pathol. 42, 405-426 (2005).</li><li>Adzemovic, M. Z., 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=Expression+of+Ccl11+associates+with+immune+response+modulation+and+protection+against+neuroinflammation+in+rats.">Expression of Ccl11 associates with immune response modulation and protection against neuroinflammation in rats.</a> PloS one. 7, 39794 (2012).</li><li>Bauer, J., 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=Endoplasmic+reticulum+stress+in+PLP-overexpressing+transgenic+rats:+gray+matter+oligodendrocytes+are+more+vulnerable+than+white+matter+oligodendrocytes.">Endoplasmic reticulum stress in PLP-overexpressing transgenic rats: gray matter oligodendrocytes are more vulnerable than white matter oligodendrocytes.</a> Journal of neuropathology and experimental neurology. 61, 12-22 (2002).</li><li>Bradl, M., Bauer, J., Flugel, A., Wekerle, H., Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+contribution+of+CD4+and+CD8+T+lymphocytes+to+T-cell+infiltration+of+the+intact+and+the+degenerative+spinal+cord.">Complementary contribution of CD4 and CD8 T lymphocytes to T-cell infiltration of the intact and the degenerative spinal cord.</a> The American journal of pathology. 166, 1441-1450 (2005).</li><li>Zhang, C., Lam, T. T., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+populations+of+microglia/macrophages+in+the+retina+and+their+activation+after+retinal+ischemia+and+reperfusion+injury.">Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury.</a> Experimental eye research. 81, 700-709 (2005).</li><li>Hirasawa, T., 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=Visualization+of+microglia+in+living+tissues+using+Iba1-EGFP+transgenic+mice.">Visualization of microglia in living tissues using Iba1-EGFP transgenic mice.</a> Journal of neuroscience research. 81, 357-362 (2005).</li><li>Norment, A. M., Salter, R. D., Parham, P., Engelhard, V. H., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+adhesion+mediated+by+CD8+and+MHC+class+I+molecules.">Cell-cell adhesion mediated by CD8 and MHC class I molecules.</a> Nature. 336, 79-81 (1988).</li><li>Hoetelmans, R. W., van Slooten, H. J., Keijzer, R., Jvan de Velde, C. J., van Dierendonck, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+formaldehyde+fixation+irreversibly+reduces+immunoreactivity+of+Bcl-2+in+the+nuclear+compartment+of+breast+cancer+cells,+but+not+in+the+cytoplasm.">Routine formaldehyde fixation irreversibly reduces immunoreactivity of Bcl-2 in the nuclear compartment of breast cancer cells, but not in the cytoplasm.</a> Applied immunohistochemistry &amp; molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry. 9, 74-80 (2001).</li><li>Hayat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microscopy, Immunohistochemistry and antigen retrieval methods for light and electron microscopy. , (2002).</li><li>Boenisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formalin-fixed+and+heat-retrieved+tissue+antigens:+a+comparison+of+their+immunoreactivity+in+experimental+antibody+diluents.">Formalin-fixed and heat-retrieved tissue antigens: a comparison of their immunoreactivity in experimental antibody diluents.</a> Applied immunohistochemistry &amp; molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry. 9, 176-179 (2001).</li><li>Shi, S. R., Key, M. E., Kalra, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+in+formalin-fixed,+paraffin-embedded+tissues:+an+enhancement+method+for+immunohistochemical+staining+based+on+microwave+oven+heating+of+tissue+sections.">Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 39, 741-748 (1991).</li><li>Huang, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+demonstration+of+hepatitis+B+core+and+surface+antigens+in+paraffin+sections.">Immunohistochemical demonstration of hepatitis B core and surface antigens in paraffin sections.</a> Laboratory investigation; a journal of technical methods and pathology. 33, 88-95 (1975).</li><li>Shi, S. R., Cote, R. J., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+immunohistochemistry:+past,+present,+and+future.">Antigen retrieval immunohistochemistry: past, present, and future.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 45, 327-343 (1997).</li><li>Taylor, C. R., Shi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval:+call+for+a+return+to+first+principles.">Antigen retrieval: call for a return to first principles.</a> Applied immunohistochemistry &amp; molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry. 8, 173-174 (2000).</li><li>Kitamoto, T., Ogomori, K., Tateishi, J., Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formic+acid+pretreatment+enhances+immunostaining+of+cerebral+and+systemic+amyloids.">Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids.</a> Laboratory investigation; a journal of technical methods and pathology. 57, 230-236 (1987).</li><li>Nelson, P. 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=Monoclonal+antibodies.">Monoclonal antibodies.</a> Molecular pathology : MP. 53, 111-117 (2000).</li><li>Van der Loos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunoenzyme multiple staining methods. , (1999).</li><li>Elias, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunohistopathology. A practical approach to diagnosis. , (2003).</li><li>Straus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Letter:+Cleavage+of+heme+from+horseradish+peroxidase+by+methanol+with+inhibition+of+enzymic+activity.">Letter: Cleavage of heme from horseradish peroxidase by methanol with inhibition of enzymic activity.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 22, 908-911 (1974).</li><li>Streefkerk, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+erythrocyte+pseudoperoxidase+activity+by+treatment+with+hydrogen+peroxide+following+methanol.">Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 20, 829-831 (1972).</li><li>Ponder, B. A., Wilkinson, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+endogenous+tissue+alkaline+phosphatase+with+the+use+of+alkaline+phosphatase+conjugates+in+immunohistochemistry.">Inhibition of endogenous tissue alkaline phosphatase with the use of alkaline phosphatase conjugates in immunohistochemistry.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 29, 981-984 (1981).</li><li>Petrelli, F., Coderoni, S., Moretti, P., Paparelli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+biotin+on+phosphorylation,+acetylation,+methylation+of+rat+liver+histones.">Effect of biotin on phosphorylation, acetylation, methylation of rat liver histones.</a> Molecular biology reports. 4, 87-92 (1978).</li><li>Yagi, T., Terada, N., Baba, T., Ohno, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+endogenous+biotin-containing+proteins+in+mouse+Bergmann+glial+cells.">Localization of endogenous biotin-containing proteins in mouse Bergmann glial cells.</a> The Histochemical journal. 34, 567-572 (2002).</li><li>Van Hecke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+Immunohistochemical+Staining+Today:+Choices+to+Make,+Challenges+to+Take.">Routine Immunohistochemical Staining Today: Choices to Make, Challenges to Take.</a> Journal of Histotechnology. 1, 45-54 (2002).</li></ol>

The authors declare no conflict of interest.

표준 IHC 절차는 종종 일반적으로 폭 넓은 경험뿐만 아니라 &quot;시행 착오&quot;접근 방식을 의미 최적의 결과를 얻기 위해 특정 조정을 필요로한다. 티슈 제조에서 대상 가시화 될 때까지 상기 프로토콜 거의 각 단계는 개별적으로 최종 결과를 개선하기 위해 수정 설계를 실시 할 수있다. 여기에 제시된 이중 염색 프로토콜은 특히 신경 염증에 영향을 포르말린 고정 파라핀 포매 쥐 CNS 및 LN 조직 섹...

methylome, 사체 또는 프로테옴 수준에서 수행 분석 고급 높은 처리량의 사용에도 불구하고, 면역 염색은 조직 샘플, 세포 배양에 직접 단백질 검출 또는 세포 도말 검사의 황금 표준 남아있다. 지역화 / 배광 패턴을 계시하여 면역 조직 화학 (IHC)의 상대적인 비율과 목표 단백질의 지형적 상호 관계를 평가하고, 그들의 생물학적 활성을 나타낼 수있다. 따라서, IHC 널리 임상 연구 및 진단 목적, 예를 들면, 치료 평가 질병 메커니즘 동물 모델에서 기능적 표현형 변화 등의 연구에 활용 본질적 조직학, 병리학, 생화학 및 면역학을 포함 IHC 크게 형광 표지 된 항체는 감염된 조직 1 폐렴 구균 항원을 식별하기 위해 처음에 사용 된 1941 년 이후로 진행되었다. V휴대 제품과 IHC에 의해 구성 요소의 isualization은 특정 항원은 (Ag)에 대한 항체 (ABS)의 결합을 기반으로합니다. 형광 태그 된 항체를 사용하는 외에, 면역 반응은 2,3- 옥시다아제 또는 알칼리 포스 파타 아제와 같은 효소 (4)을 이용하여 가시화 될 수있다. 방사능 라벨자가 방사선 시각화하는 동안 또한, 콜로이드 금 항체 태그 (5)는 광학 및 전자 현미경 모두에 의해 특정 항원 항체 반응을 검출하기 위해 사용된다. 용 Ag-Ab의의 면역 직접 및 간접적 인 방법을 통해 검출 할 수있다. 직접적인 방법은 직접 차 애비 6 레이블 사용하기 때문에, 본질적으로 빠르고 간단합니다. 그러나, 감도 부족으로 심각한 간접 방법이 직접 사람에게 바람직하다. 두 단계 간접 검출 방법은 상기 제 7 층과 같은 기본 복근 향한 이차 복근 첫번째로, 표지되지 않은 기본 복근 및 표지 필요로한다.신호 증폭을 더 포함함으로써 달성 될 수 있으며, 효소 - 결합 급 순이 (세 단계 간접법) 보조 AB와 결합하는 것이다. 일반적으로 사용되는 간접 검출 방법은 아비딘 - 비오틴과 퍼 옥시 다제 - antiperoxidase (PAP)입니다. 대안 적으로, 알칼리성 포스파타제 antialkaline 포스파타제 (APAAP) 복합체 대신 PAP 방법을 사용할 수있다. 특히, 알칼리성 포스파타제 (AP) 방법은 immunoperoxidase 방법 4보다 더 민감한 것으로 나타납니다. 아비딘 - 비오틴 복합체 (ABC) 방법은 표지 아비딘 - 비오틴 복합체 (LAB) 중 하나와 함께 바이오틴 차 순이를 사용, 또는 스트렙 타비 딘 - 비오틴 복합체 (SLAB) 표지. 검출 감도는 상기 퍼 옥시다아제 또는 알칼리 포스 파타 아제 표지 아비딘 (8)를 포함함으로써 증가 될 수있다. 사용의 다른 검출 방법은 고분자 라벨, 타이 라민 증폭 및 면역 구름 원 9입니다. 특히, 다른 검출 방법은 동일한 조직에 다수의 Ag 검출을 위해 결합 될 수있다여기에 제시된 1978 4. 동시 이중 면역 염색에 처음보고 된 샘플은 각각, 퍼 옥시 다제 - 결합 및 AP 접합 이차 항체를 이용하여 포르말린 고정 된 파라핀 포매 쥐 CNS 및 LN 조직 절편에서 수행 하였다. 신호는 각각 (FB) APAAP 복잡한 3,3&#39;- diaminobencidine (DAB) 색 원체 및 패스트 블루를 사용하여 시각화 하였다.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Isoflurane (Isofluran): 1-Chlor-2,2,2-trifluorethyl (difluormethyl ether) 2-Chlor-2-(difluormethoxy)-1,1,1-trifluorethan (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;ClF&lt;sub&gt;5&lt;/sub&gt;O)</td><td>Baxter</td><td>1001936060</td><td>Eye irradiation. Probably influences fertility and damages baby in uterus. Administer only in adequately equipped anesthetizing environment.</td></tr><tr><td>Sodiumchloride (NaCl)</td><td>Merck</td><td>1.0604</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS), tablet</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>Paraformaldehyde (OH(CH&lt;sub&gt;2&lt;/sub&gt;O)&lt;sub&gt;n&lt;/sub&gt;H (n = 8 - 100)), 4% in 1x PBS</td><td>Apl pharma</td><td>34 24 28</td><td>Health hazard. Corrosive. Flamable. Acute toxicity.</td></tr><tr><td>Ethanol &amp;#8805;99.5%, absolute (CH&lt;sub&gt;3&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;OH)</td><td>Sigma-Aldrich</td><td>459844-1L</td><td>Flamable.</td></tr><tr><td>Xylene (Xylenes, histological grade; C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;(CH&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt; </td><td>Sigma-Aldrich</td><td>534056</td><td>Flamable. Acute toxicity.</td></tr><tr><td>Histo-Comp Paraffin Wax</td><td>Tissue-Tek</td><td>V0-5-1001</td><td></td></tr><tr><td>Adhesive Microscope Slides</td><td>Starfrost</td><td>MIC-1040-W</td><td></td></tr><tr><td>Xylol substitute XEM-200 </td><td>Vogel GmbH</td><td>ND-HS-200</td><td>Respiratory sensitization. Carcinogenicity.</td></tr><tr><td>&amp;alpha;-CD8 (Ox-8) mouse anti-rat, primary antibody</td><td>AbD Serotec</td><td>MCA48G</td><td></td></tr><tr><td>&amp;alpha;-Iba1 (AIF1) mouse anti-rat, primary antibody</td><td>Millipore</td><td>MABN92</td><td></td></tr><tr><td>&amp;alpha;-CD68 (ED1) mouse anti-rat, primary antibody</td><td>AbD Serotec</td><td>MCA341R</td><td></td></tr><tr><td>&amp;alpha;-eotaxin C-19 (CCL11) goat anti-rat, primary antibody</td><td>Santa Cruz BT</td><td>SC-6181</td><td></td></tr><tr><td>Alkaline phosphatase (AP)-conjugated secondary antibody</td><td>Dakopatts, Denmark</td><td>D0314</td><td></td></tr><tr><td>Biotinylated secondary antibody</td><td>Amersham Biotech</td><td>RPN1025</td><td></td></tr><tr><td>Avidin- horseradish peroxidase complex (HRP) </td><td>Sigma-Aldrich</td><td>A3151</td><td></td></tr><tr><td>Naphthol AS-MX phosphate (C&lt;sub&gt;19&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;NO&lt;sub&gt;5&lt;/sub&gt;P)</td><td>Sigma-Aldrich</td><td>N4875-1G</td><td>Acute toxicity.</td></tr><tr><td>Fast Blue RR Salt, Azoic Diazo No. 24 (C&lt;sub&gt;15&lt;/sub&gt;H&lt;sub&gt;14&lt;/sub&gt;ClN&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt; x 1/2 ZnCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>FBS25</td><td></td></tr><tr><td>Levamisol hydrochloride (C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;13&lt;/sub&gt;ClN&lt;sub&gt;2S&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>31742</td><td>Acute toxicity.</td></tr><tr><td>3,3&#039;-Diaminobenzidine tetrahydrochloride (DAB Chromogen)</td><td>DAKO</td><td>S3000</td><td>Highly flammable. Toxic.</td></tr><tr><td>Copper sulphate (CuSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Merck</td><td>1.02791</td><td>Acute toxicity. Environmental hazzard.</td></tr><tr><td>GelTol Aqueous Mounting Medium</td><td>Thermo Electron Corporation</td><td>230100</td><td></td></tr><tr><td>Hydrogen peroxide, 30% (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Merck</td><td>107210</td><td>Acute toxicity.</td></tr><tr><td>Methanol (CH&lt;sub&gt;3&lt;/sub&gt;OH)</td><td>Fluka</td><td>65543</td><td>Acute toxicity. Respiratory sensitization. Carcinogenicity. Flammable.</td></tr><tr><td>Tris (hydroxymethyl) aminomethane, TRIS base (C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt; )</td><td>AppliChem</td><td>A1379</td><td>Skin and eye irritation.</td></tr><tr><td>Tris Buffered Saline (TBS), tablet</td><td>Sigma-Aldrich</td><td>T5030</td><td></td></tr><tr><td>Di- Sodium hydrogen phosphate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Merck</td><td>1.0658</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; x 1H&lt;sub&gt;2&lt;/sub&gt;O) </td><td>Merck</td><td>1.06346</td><td></td></tr><tr><td>Fetal calf serum (FCS) </td><td>Cambrex BioScience</td><td>DE-14-802F</td><td></td></tr><tr><td>DAKO cytomation wash buffer 10x </td><td>DAKO</td><td>S3006</td><td>Should be stored at 2-8 &amp;deg;C to inhibit bacterial growth. Avoid foaming.</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide; DMF (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;7&lt;/sub&gt;NO)</td><td>Fluka</td><td>40250</td><td>Flammable. Acute toxicity.</td></tr><tr><td>Glas coverslips 24 x 36 mm </td><td>Menzel-Gl&amp;#228;ser</td><td>BB024036A1</td><td></td></tr><tr><td>Hydrochloric acid, conc. (HCl)</td><td>Sigma-Aldrich</td><td>30721</td><td>Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic.</td></tr><tr><td>Hydrochloric acid solution volumetric, 2 M HCl (2 N)</td><td>Fluka</td><td>71826</td><td>Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic.</td></tr><tr><td>Sodium nitrite, ReagentPlus, &amp;#8805;99.0% (NaNO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S2252</td><td>Oxidant. Toxic. Dangerous for the environment.</td></tr><tr><td>Ethylenedinitrilotetraacetic acid disodium salt dihydrate (EDTA; C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;14&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;Na&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Merck</td><td>1.08454</td><td>Oral exposures may cause reproductive and developmental effects.</td></tr><tr><td colspan="4">&lt;strong&gt; Equipment&lt;/strong&gt;</td></tr><tr><td>Tissue-Tek&lt;sup&gt;V.I.P&lt;/sup&gt; Vacuum Infiltration Processor</td><td>Sakura</td><td>5902 VIP Jr. 115 V, 60 Hz</td><td></td></tr><tr><td>Hacker-Bright 8000 Series Base Sledge Microtome </td><td>Hacker instruments</td><td></td><td></td></tr><tr><td>Household food steamer </td><td>Braun</td><td>MultiGourmet FS 20</td><td></td></tr><tr><td>Light microscope </td><td>Leica Polyvar 2</td><td></td><td></td></tr></tbody></table>

immunohistochemical analysis in the rat central nervous system and peripheral lymph node tissue sections

면역 조직 화학 (IHC)이 높은 특정 안정적이고 매력적인 단백질의 시각화를 제공합니다. IHC 기반의 빛깔 표지의 정확한 성능과 해석은 중추 신경계 (CNS) 등의 지방 함량이 높은 조직에서 목적 단백질 사이의 상호 관계를 평가하기 위해 사용될 때 특히 도전적이다. 우리의 프로토콜은 특히 쥐 CNS와 신경 염증에 의해 영향을받는 주변 림프절 (LN) 모두 구조 및 가용성 단백질의 검출을 위해 조정 된 표준 immunolabeling 기술의 정교화를 나타냅니다. 그럼에도 불구하고, 추가 수정하거나하지 않고, 우리의 프로토콜 가능성도 여기에 제시된 것보다 다른 기관과 종, 기타 관련 단백질 표적의 탐지를 위해 사용될 수있다.

더블 immunostainings (공동의 염색)는 포르말린 고정 파라핀 포함 된 쥐 CNS 및 LN 절에서 수행되었다. 3-5 μm의 두께 조직 슬라이스 사전 코팅 접착 유리 슬라이드에 연속적으로 설치 이전에 10,11,12 설명으로 처리, 썰매 마이크로톰을 사용하여 절단 하였다. 간단히 deparaffinizing, 조직 재수 내생 퍼 옥시 다제 불 활성화 한 후, 절편을 비특이적 결합 부위를 제거하는 단계를...

Watch this Scientific Journal Video about Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections at JoVE.com

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

쥐 중추 신경계에서 면역 조직 화학 분석 및 주변 림프절 조직 섹션

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

immunohistochemical-analysis-rat-central-nervous-system-peripheral

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.0K 조회수.  Karolinska Institutet. We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

비디오: Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

림프절 저온 절에서 종양 세포 접착의 정량화

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...

연구

암 연구학

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

미세 아교 세포와 신경 세포에 대한 예 염색 : 저온 절단 된 쥐의 뇌 조직에서 면역 조직 화학에 대한 입문서

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

신경과학

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

신경 화학적 마커에 대한 Biocytin 충전 및 가공 섹션의 면역 염색

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

실험적인 자가면역 뇌척수염 중 중추신경계에서 림프구 침투의 흐름 세포 분석

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

면역학 및 감염병학

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

면역 세포의 다운스트림 분석을 위한 중추신경계 조직 및 관련 수막 분리

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

근 적외선 형광 및 고 분해능 주사를 사용 하 여 설치류 뇌의 단백질 발현 측정

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To maintain the morphology of these delicate tissues, it is routine to administer a chemical fixative such as paraformaldehyde via cannulation of the heart in anesthetized animals (transcardial perfusion). Transcardial perfusion of the mouse heart has traditionally relied on the use of peristaltic pumps or air pressure to deliver both the saline and fixative solutions necessary for this process. As an easily accessible alternative to these methods, this work demonstrates the use of a gravity-fed method of perfusate delivery that uses materials available in most hardware stores. 
To validate this new perfusion method, this work demonstrates all the subsequent steps necessary for the sensitive detection of phosphorylated &#945;-synuclein in both the brain and spinal cord. Included in these steps are the dissection of the fixed brain and spinal cord tissues, rapid freezing/embedding and cryosectioning of the tissues, and immunofluorescent staining. As this method results in whole-body delivery of the fixative, it may also be used to prepare other non-neuronal tissues for histologic analysis.

A convenient gravity-fed perfusion method for histological analysis of the mouse central nervous system is presented. Immunofluorescent detection of phosphorylated &#945;-synuclein is demonstrated in a mouse model of Parkinson's disease. This work also comprehensively describes the transcardial perfusion, dissection, tissue freezing/embedding, and frozen sectioning steps.

마우스 중추 신경계의 조직학적 분석을 위한 중력 공급 심질 관류 방법

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To ...

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

자유 부유 조직 절편을 사용한 조직학적 기반 염색

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

생물학

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Source: Singh, P., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/60405">Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020)
...