Ö Stephens (University of Bristol, Bristol, İngiltere) TPST1-EGFP DNA plazmid yanı sıra için Lakshmi Narasimhan Govindarajan yazılım optimizasyonu ile yardımcı olduğunuz için teşekkür etmek istiyorum. Bu eser L.L. Ulusal Tıbbi Araştırma Konseyi (NMRC/CBRG/007/2012), Milli Eğitim Bakanlığı (AcRF Tier1 RG 18/11, RG 48/13 ve RG132/15 ve AcRF Tier2 MOE2015-T2-2-073) hibe tarafından desteklenmiştir

<ol>
	<li>Glick, B. S., Luini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+Golgi+traffic:+a+critical+assessment.">Models for Golgi traffic: a critical assessment.</a> Cold Spring Harb Perspect Biol. 3 (11), 005215 (2011).</li><li>Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+mammalian+Golgi.">Architecture of the mammalian Golgi.</a> Cold Spring Harb Perspect Biol. 3 (7), (2011).</li><li>Lu, L., Hong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+endosomes+to+the+trans-Golgi+network.">From endosomes to the trans-Golgi network.</a> Semin Cell Dev Biol. , (2014).</li><li>De Matteis, M. A., Luini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exiting+the+Golgi+complex.">Exiting the Golgi complex.</a> Nat Rev Mol Cell Biol. 9 (4), 273-284 (2008).</li><li>Lu, L., Ladinsky, M. S., Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisternal+organization+of+the+endoplasmic+reticulum+during+mitosis.">Cisternal organization of the endoplasmic reticulum during mitosis.</a> Mol Biol Cell. 20 (15), 3471-3480 (2009).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> J Cell Biol. 190 (2), 165-175 (2010).</li><li>Tie, H. C., 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=A+novel+imaging+method+for+quantitative+Golgi+localization+reveals+differential+intra-Golgi+trafficking+of+secretory+cargoes.">A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes.</a> Mol Biol Cell. 27 (5), 848-861 (2016).</li><li>Trucco, 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=Secretory+traffic+triggers+the+formation+of+tubular+continuities+across+Golgi+sub-compartments.">Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments.</a> Nat Cell Biol. 6 (11), 1071-1081 (2004).</li><li>Cole, N. B., Sciaky, N., Marotta, A., Song, J., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi+dispersal+during+microtubule+disruption:+regeneration+of+Golgi+stacks+at+peripheral+endoplasmic+reticulum+exit+sites.">Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.</a> Mol Biol Cell. 7 (4), 631-650 (1996).</li><li>Rogalski, A. A., Bergmann, J. E., Singer, S. J. <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+microtubule+assembly+status+on+the+intracellular+processing+and+surface+expression+of+an+integral+protein+of+the+plasma+membrane.">Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.</a> J Cell Biol. 99 (3), 1101-1109 (1984).</li><li>Van De Moortele, S., Picart, R., Tixier-Vidal, A., Tougard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nocodazole+and+taxol+affect+subcellular+compartments+but+not+secretory+activity+of+GH3B6+prolactin+cells.">Nocodazole and taxol affect subcellular compartments but not secretory activity of GH3B6 prolactin cells.</a> Eur J Cell Biol. 60 (2), 217-227 (1993).</li><li>Nakamura, 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=Characterization+of+a+cis-Golgi+matrix+protein,+GM130.">Characterization of a cis-Golgi matrix protein, GM130.</a> J Cell Biol. 131, 1715-1726 (1995).</li><li>Roth, J., Berger, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+localization+of+galactosyltransferase+in+HeLa+cells:+codistribution+with+thiamine+pyrophosphatase+in+trans-Golgi+cisternae.">Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.</a> J Cell Biol. 93 (1), 223-229 (1982).</li><li>Baeuerle, P. A., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+sulfation+is+a+trans-Golgi-specific+protein+modification.">Tyrosine sulfation is a trans-Golgi-specific protein modification.</a> J Cell Biol. 105 (6), 2655-2664 (1987).</li><li>Honda, A., Al-Awar, O. S., Hay, J. C., Donaldson, 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=Targeting+of+Arf-1+to+the+early+Golgi+by+membrin,+an+ER-Golgi+SNARE.">Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE.</a> J Cell Biol. 168 (7), 1039-1051 (2005).</li><li>Lavieu, G., Zheng, H., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stapled+Golgi+cisternae+remain+in+place+as+cargo+passes+through+the+stack.">Stapled Golgi cisternae remain in place as cargo passes through the stack.</a> Elife. 2, 00558 (2013).</li><li>Zhao, X., Lasell, T. K., Melancon, P. <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+large+ADP-ribosylation+factor-guanine+nucleotide+exchange+factors+to+different+Golgi+compartments:+evidence+for+distinct+functions+in+protein+traffic.">Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic.</a> Mol Biol Cell. 13 (1), 119-133 (2002).</li><li>Dejgaard, S. Y., Murshid, A., Dee, K. M., Presley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy-based+linescan+methodologies+for+intra-Golgi+localization+of+proteins.">Confocal microscopy-based linescan methodologies for intra-Golgi localization of proteins.</a> J Histochem Cytochem. 55 (7), 709-719 (2007).</li><li>Fourriere, L., Divoux, S., Roceri, M., Perez, F., Boncompain, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-independent+secretion+requires+functional+maturation+of+Golgi+elements.">Microtubule-independent secretion requires functional maturation of Golgi elements.</a> J Cell Sci. 129 (17), 3238-3250 (2016).</li><li>Volchuk, 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=Countercurrent+distribution+of+two+distinct+SNARE+complexes+mediating+transport+within+the+Golgi+stack.">Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack.</a> Mol Biol Cell. 15 (4), 1506-1518 (2004).</li><li>Popoff, V., Adolf, F., Brugger, B., Wieland, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+budding+within+the+Golgi+stack.">COPI budding within the Golgi stack.</a> Cold Spring Harb Perspect Biol. 3 (11), 005231 (2011).</li></ol>

Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Daha önce yerelleştirme ışık mikroskobu altında Golgi protein ağırlıklı olarak korelasyon veya protein görüntünün Golgi işaret bilinen Yerelleştirme15,16resimle örtüşen tarafından sayılabilir, 17. Elde edilen korelasyon veya örtüşen katsayısı ne kadar yakın test Golgi işaretçisi için dağınık şekilde proteindir yansıtır. Bu yaklaşım için en az üç uyarılar vardır. İlk olarak, korelasyon...

Golgi kompleksi memeli hücreleri1,2,3' te proteinler ve yağlar (bundan sonra yükler) salgı/endositik kaçakçılığı önemli rol oynar. Golgi yükler sadece için çeşitli alt hücresel kompartmanlarda sıralanmış ama aynı zamanda glikozilasyon çeşitli türlerine göre değişiklik. Memeli Golgi kompleksi çok sayıda yanal bağlı Golgi yığınlar, ki genellikle 4-11 sıkı bitişik ve düz zar kesesi cisternae denilen oluşur oluşur. Seri olarak yığılmış Golgi cisternae daha fazla, diğer bir ucundan CIS, medial ve transkategorize edilir-cisternae. Trans-yan Golgi yığın, trans- transolarak adlandırılan borulu ve retikulum membran ağın çoğu membran sac geliştirir-Golgi ağ (TGN)4. Salgı yol endoplazmik retikulum (ER) türetilmiş yükler, CIS, Golgi yığın girin-yan ve daha sonra sırayla medial ve transyoluyla geçmek-cisternae. Gönderileri sonunda Golgi trans, çıkmak-Golgi veya TGN plazma zarı, endosomes veya salgı granül destining.
Nasıl yükler Golgi yığın transit ve Golgi cisternal kuruluşu nasıl saklandığına ilişkin moleküler ve hücresel mekanizmaları gizemli kalır ve hala bir hararetli tartışmalara1altında şu anda. Bu alandaki zorluklardan biri Golgi cisternae bir optik mikroskobu çözünürlük beri sadece elektron mikroskobu (EM) altında çözülebilir (~ 200 nm) bireysel Golgi cisternae (< 100 nm her iki cisternal kalınlıkta gidermek için yeterli değil ve mesafe) Bu nedenle, alt-Golgi yerelleştirme ikamet proteinlerin ve transit gönderileri geleneksel belirlenir tarafından IMMUNO-altın EM. Ancak, IMMUNO-altın EM çok teknik olarak talep ediyor ve çoğu hücre biyolojisi laboratuvarları yeteneği. EM çözünürlük-ebilmek var olmak alt nanometre, IMMUNO-altın EM tarafından tanınan çözünürlüğü büyük ölçüde karmaşık antikor (birincil ve ikincil antikor) boyutunu ve altın parçacık tarafından engel oluyor ve 20 kötü olabilir rağmen nm. Ayrıca, EM görüntüleri 2D ince-bölümler yerine bağlı olarak göreceli konumunu ve yönünü 2D bölüm5hatalı sonuçlara yol açabilir Golgi 3D bir genel görünümünü elde edilir. Örneğin, bir EM tek bölüm okuyan ondan beri her ikisi de aynı yuvarlak membran profiller görüntüleyebilirsiniz güvenilir bir tübül ortogonal görünümünden bir vezikül ayırt değiştiremiyor. Emisyon tükenmesi (STED), photoactivated yerelleştirme mikroskobu (PALM) ve Stokastik optik imar mikroskobu (3D yapılı aydınlatma mikroskobu (3D-SIM), gibi süper çözümleme mikroskobu teknikleri son gelişiyle uyarılmış Fırtına), alt-Golgi yapıları ışık mikroskoplar6altında çözmek sağlar. Ancak, önemli ölçüde kullanımları hücre biyolojik çalışmada, Golgi sınırlayabilirsiniz en az dört dezavantajları vardır. 1) geçerli süper çözümleme teknikleri çoğu hücre biyolojisi laboratuvarları olan pahalı ve özel donanım yapılandırması gerektirir. 2) bazı süper çözümleme teknikleri için özel Floresans etiketleme iletişim kurallarına gerek. 3) rağmen en iyi koşul altında bu tekniklerin 20-110 nm mekansal çözünürlükte iddia, gerçek örneklerinde elde pratik çözünürlük çok daha kötü olabilir. 4) içinde karşılaştırma için geleneksel mikroskobu, bu süper çözümleme teknikleri hala yürütülmesi konusunda zorluklar çok renkli, 3D veya hücre Imaging, tek başına veya birlikte yaşamak. Muhtemelen en önemlisi IMMUNO-altın EM ve süper çözümleme mikroskobu teknikleri nitel nicel yerelleştirme veri yerine verim.
Kısmen yukarıda belirtilen sorunları çözmek çalışırken, biz son zamanlarda Golgi protein yerelleştirme merkezleri kütlenin (ışık), sistematik ve kantitatif bir Golgi yerelleştirmek için Imaging tarafından adlı bir konvansiyonel ışık mikroskobu dayalı yöntem geliştirdik IMMUNO-altın EM7eşdeğer bir çözünürlükte protein. Bu yöntemde, kültürlü memeli hücrelerinde Golgi Golgi mini-yığınlar nocodazole, bir Mikrotubul depolymerizing ilaç tedavisi tarafından dağınık olduğunu. Nocodazole kaynaklı Golgi mini yığınlarının (bundan sonra Golgi mini-yığınlar) yakından organizasyon ve hücresel işlevler8,9,10yerli Golgi yığınlarda benzer kapsamlı çalışmalar göstermiştir, 11. Bir test protein yerelleştirme sayının (LQ) ışık elde edilebilir ve nicel alt-Golgi yerelleştirme gösterir. LQs sayısal değerler karşılaştırılabilir ve 25'ten fazla Golgi işaretlerinin LQ veritabanı mevcut olmuştur.
IŞIK içinde endojen veya exogenously ifade GM130, GalT-mCherry ve test protein (x) tarafından üç kez etiketli Golgi mini-yığınlar. GM130 ve GalT-mCherry, CIS- ve trans-Golgi işaretleri sırasıyla12,13, referans noktaları sağlamak. Üçlü Floresan, kırmızı (R), yeşil (G) ve far-red (B), yapay olarak kırmızı, yeşil ve mavi sırasıyla görüntülenir. Merkezi Floresans kitle (bundan sonra Merkezi) alt piksel çözünürlük elde etmek için kabul edilir. Golgi eksen GalT-mCherry olan için GM130 merkezi üzerinden vektör olarak tanımlanır. Golgi Mini yığın Golgi ekseni etrafında sonsuz dönme simetri ile silindirik yapısı olarak modellenmiştir. Bu nedenle, Golgi Mini yığını daha fazla Golgi eksen boyunca tek boyutlu bir yapı olarak modellenebilir. LQ testi protein x dx/d1dx olduğu x merkezine uzaklik bu GM130, d1 GM130 olan için merkezi GalT-mCherry dan mesafe olsa, olarak tanımlanır. X Merkezi Eksen dışı projeksiyon Aksiyel uzaklığı hesaplama için kullanılır. Golgi eksen, Aksiyel açı, dx, d1, açı α ve β açısı, ışık için de dahil olmak üzere değişkenler, şematik Resim 1' de gösterilmiştir. LQ Golgi Aksiyel açısı bağımsız olsa da Golgi mini-yığınlar rastgele bir hücreye yönlendirmek.
Golgi mini-yığınlar Albümdeki inhomogeneous görünür. Analyzable Golgi mini-yığınlar ışık için seçmek için üç ölçüt geliştirdik. 1) sinyal-gürültü oranı kriteri, hangi ≥ 30 her kanaldaki arka plan standart sapma (SD) Golgi Mini yığıta toplam yoğunluğunu oranıdır. Bu ölçüt Merkezi kütlenin, Golgi mini-yığınlar sinyal noise oranları üzerinde bağlıdır konumlandırma doğruluğunu sağlamaktır. 2) d1≥ 70 gerektirir Aksiyel açı ya da mesafe kriteri, nm. d1 Golgi Aksiyel açısı artış ile azalır. Ne zaman Aksiyel açı 90 ° yaklaşıyor ya da dikey, Mini yığını d1 sigara çözülebilir olur 0 yaklaşıyor. d1≥ 70 nm etkili bir şekilde dikey Golgi mini-yığınlar hariç. 3) co-doğrusallık ölçütü, hangi da | tan α | veya | tan β | ≤ 0,3 olduğunu. Bu ölçüt mini bir yığın üç Merkezi yeterince co-doğrusal Golgi Mini yığını için bizim tek boyutlu model olmasını sağlar. Tüm ışık mikroskoplar renk sapması olan kırmızı, yeşil ve far-red floresan merkezleri göreli konumları ciddi deforme edebilirsiniz acı. Renk sapmaları mikroskop sistemlerinin deneysel olarak kırmızı, yeşil ve far-red floresan tarafından üç kez etiketli 110 nm boncuk Imaging tarafından kalibre edilmiş. Her boncuk görüntüsünün kırmızı merkezi boncuk gerçek konumunu tanımlanır ve Kromatik vardiya-yeşil ve far-red merkezleri tarafından birinci dereceden polinom fonksiyonların donatılmıştır. Golgi mini-yığınlar merkezlerinden Kromatik yeşil ve far-red Kanallar olarak kaydırır düzeltmek için polinom fonksiyonların tabi.
IŞIK ile a kararlılık-in elde edebilirsiniz ~ 30 nm standart koşullar altında Golgi eksen boyunca. Önemlisi, kantitatif Golgi protein var eşlemek için uyguladıkları bir yöntem sağlar. IŞIK geleneksel mikroskoplar, geniş alanlı gibi veya confocal mikroskoplar, ortak Floresans etiketleme iletişim kuralları kullanılarak gerçekleştirilebilir. Görüntüleme ve veri işleme gibi bir saat gibi kısa sürebilir. IŞIK, biz doğrudan salgı kargo aşamalı geçiş CIS- transiçin göstermiştir-Golgi7yan.

<table>
	<tbody>
		<tr>
			<td class="xl67">fluorescence beads. Commercial name: TetraSpeck beads</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">T7279</td>
			<td class="xl67">As multi-color beads to calibrate chromatic-shift of the microscope.</td>
		</tr>
		<tr>
			<td class="xl67">Glass coverslip &#934; 12 mm (No. 1.5)</td>
			<td class="xl67">Menzel</td>
			<td class="xl75">CB00120RAC</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Glass coverslip &#934; 25 mm (No. 1.5)</td>
			<td class="xl67">Menzel</td>
			<td class="xl75"></td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">DMEM</td>
			<td class="xl67">Capricon</td>
			<td class="xl75">DMEM-HPA-P50</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Trypsin-EDTA</td>
			<td class="xl67"></td>
			<td class="xl75"></td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">FBS</td>
			<td class="xl67">GE Hyclone</td>
			<td class="xl75">SV30160.03</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Nocodazole</td>
			<td class="xl67">Merck</td>
			<td class="xl75">487928</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">transfection reagent. Commercial name: Lipofectamine 2000</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">11668-019</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">transfection medium. Commercial name: OptiMEM</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">31985070</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">TPST1-EGFP</td>
			<td class="xl67">Addgene</td>
			<td class="xl75">66617</td>
			<td class="xl67">A gift from D. Stephens (University of Bristol, Bristol, United Kingdom)</td>
		</tr>
		<tr>
			<td class="xl67">GalT-mCherry</td>
			<td class="xl67"></td>
			<td class="xl75"></td>
			<td class="xl67">Made in our lab.</td>
		</tr>
		<tr>
			<td class="xl67">paraformaldehyde</td>
			<td class="xl67">Merck</td>
			<td class="xl75">1.04005.1000</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">saponin</td>
			<td class="xl67">Sigma-Aldrich</td>
			<td class="xl75">47036</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">poly(vinyl alcohol) (Mw ~31,000). Commercial name: Mowiol-488</td>
			<td class="xl67">CALBIOCHEM</td>
			<td class="xl75">475904</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">BSA</td>
			<td class="xl67">Sigma-Aldrich</td>
			<td class="xl75">A9647</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Mouse anti-GM130</td>
			<td class="xl67">BD Biosciences</td>
			<td class="xl75">610823</td>
			<td class="xl67">Primary antibody for human GM130</td>
		</tr>
		<tr>
			<td class="xl67">far-red fluorescence conjugated goat anti-mouse IgG. Commercial name: Alexa Fluor 647 conjugated goat anti-mouse IgG</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">A-21235</td>
			<td class="xl67">Far red fluorescence conjugated secondary antibody</td>
		</tr>
	</tbody>
</table>

quantitative localization of a golgi protein by imaging its center of fluorescence mass

CISdahil olmak üzere alt-Golgi bölgelere daha fazla sınıflandırılabilir seri olarak yığılmış membran cisternae Golgi kompleksi oluşur-Golgi, medial-Golgi, trans-Golgi ve trans-Golgi ağ. Golgi hücresel işlevlerini kendi ikamet proteinler karakteristik dağıtım tarafından belirlenir. Konvansiyonel ışık mikroskobu Uzaysal çözünürlük alt-Golgi yapısı veya cisternae gidermek için çok düşüktür. Böylece, IMMUNO-altın elektron mikroskopi sub-Golgi düzeyinde bir protein yerelleştirmek için seçim yöntemidir. Ancak, teknik ve araç çoğu hücre biyolojisi laboratuvarları yeteneği vardır. Biz burada Golgi protein yerelleştirme merkezleri kütlenin (ışık) sistematik ve kantitatif Golgi protein yerelleştirmek için Imaging tarafından denilen bizim son zamanlarda geliştirilen süper çözümleme yöntemi açıklanmaktadır. IŞIK standart floresan etiketleme iletişim kuralları ve geleneksel geniş-alan veya confocal mikroskoplar üzerinde temel alır. Shift renk sapmaları mikroskobik sistem, resim alma ve sonrası edinme analiz kalibrasyonu içerir. Bir test protein alt-Golgi lokalizasyonu kantitatif yerelleştirme bölüm ifade edilir. IŞIK dört ana avantajı vardır; hızlı, geleneksel yöntemler ve araçlar dayalı, yerelleştirme sonucudur nicel ve bu tanıyor ~ 30 nm pratik çözünürlük Golgi eksen boyunca. Burada bir test Golgi protein yerelleştirmek için ışık detaylı Protokolü açıklar.

Bizim laboratuarda kullanılan gibi bir planı apochromatic objektifle donatılmış modern araştırma sınıf ışık mikroskobu çok az renk sapmaları (Şekil 2A) gösterir. Ancak, dikkatli bir muayene çok renkli floresan boncuk görüntünün üst karakter aynı boncuk (Şekil 2B) farklı renk görüntülerin ortaya çıkarabilir. Biz kırmızı kanalı renk sapmaları özgür ve bu nedenle...

Watch this Scientific Journal Video about Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass at JoVE.com

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

Golgi sakinlerinin kesin yerelleştirme Golgi hücresel işlevlerini anlamak için önemlidir. Ancak, geleneksel optik mikroskobu alt-Golgi yapısı dönüştürememiş demektir. Burada iletişim kuralı için kantitatif bir protein alt-Golgi yerelleştirme belirlemek geleneksel mikroskobu dayalı süper çözümleme yöntemi açıklanmaktadır.

Floresans Merkezi kütlesi Imaging tarafından Golgi protein nicel yerelleştirme

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

quantitative-localization-golgi-protein-imaging-its-center

Research

JoVE Journal

Biology

10.7K Görüntüleme.  Nanyang Technological University. Golgi sakinlerinin kesin yerelleştirme Golgi hücresel işlevlerini anlamak için önemlidir. Ancak, geleneksel optik mikroskobu alt-Golgi yapısı dönüştürememiş demektir. Burada iletişim kuralı için kantitatif bir protein alt-Golgi yerelleştirme belirlemek geleneksel mikroskobu dayalı süper çözümleme yöntemi açıklanmaktadır.

Video: Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Biyoloji

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Yapısal Kütle Spektrometresi büyük protein kompleksleri Analiz

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Dayanarak protein-protein etkileşimleri ve kimliği için bir protokol<sup&gt; 15</sup&gt; K Metabolik Etiketleme, immünopresipitasyon, Kantitatif Kütle Spektrometrisi ve Affinity Modülasyon

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Gelişmiş 3D Floresan Mikroskobu kullanılarak Otofaji Kantitatif Analiz

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Bimoleküler Floresan tamamlama akışı Sitometrik Analizi: Protein-protein etkileşim Eğitim Bir Yüksek Verimli Kantitatif Yöntemi

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Hızlı Analiz ve Floresans Mikroskopi Görüntüler Exploration

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

Bir floresan bazlı Kalsiyum Mobilizasyon Deneyi G proteini ile birleştirilmiş alıcıların karakterizasyonu

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.