Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
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Bu çalışma Ulusal Sağlık Enstitüleri Sağırlık ve Diğer İletişim Bozuklukları Nörolojik Bozukluklar ve İnme Ulusal Enstitüsü (NINDS) ve Ulusal Enstitüsü (NIDCD), (NIH) İntramural Programları tarafından desteklendi. Biz NINDS EM tesis ve yardım için NIDCD gelişmiş görüntüleme çekirdek (kod # ZIC DC 000081-03) teşekkür ederim.

Bakım ve hayvanların taşıma NIH Hayvan Bakımı ve Kullanımı Komite Kurallarına göre idi. 12 saat ışık: Postnatal gün (P) bilateral üstün colliculus ile 1-1.2% CTB enjekte 15-21 Sprague-Dawley sıçanları, 12 muhafaza edildi karanlık döngüsü. 1. Retina Doku Fixation <ol><li> Bir diseksiyon mikroskobu, çok ince ipuçları, makas, selüloz filtre kağıdı, plastik pipet ve bir mikroskop lamı ile 2 forseps: Aşağıdaki malzemeler ve aletler monte edin. </li><li> 2.0 mi, halotan (bir uçucu anestezik) ile kapalı bir bölme sıçan anestezisi. ayak tutam veya göz kırpma refleksi eksikliği sonra arka pençe çekilmesi eksikliği: bu yöntemlerle yeterli anesthetization belirleyin. Sonra, bir giyotinle birlikte derhal başını kesmek. pH 7.4&#39;te 0.1 M fosfat tampon maddesi (PB) içinde% 4 paraformaldehid içeren bir cam tabak içinde iris makas ve yer ile gözler çıkarın. </li><li> diseksiyon mikroskobu kullanarak, mısır kaldırmakgöz küresinin ön keserek ad. forseps ile iç retina yüzeyinden lens ve vitreus çıkarın. </li><li> Retina Göz yuvası izole edilene kadar iki forseps ile sklera soyun. </li><li> Bir jilet ile 200 mikron kalınlığında şeritler ve pH-shift tespitin tabi - 100 içine hemen retina kesti. </li><li> Oda sıcaklığında 20 dakika için 10 - pH 10.5, 30 dakika ve daha sonra% 4 paraformaldehid artı% 0.01 glutaraldehid - 20 pH 6.0&#39;da 0.1 M PB içinde% 4 paraformaldehit retina şeritler düzeltildi. </li><li> (4 ° C&#39;de pH 7.4), 0.15 mM CaCI2 ile PB pek çok kez yıkamadan sonra, 0.1 (% 30 O / N sonra% 10,% 20,% 30, her 60 dakikada bir) M gliserol ile retina şeritler cryoprotect ikamesi dondurmak için önce PB. </li></ol> 2. Freeze-ikamesi NOT: Bu donma-ikame yöntemi erken yayınlanmış bir protokol 19,20 modifiye edilir. Ayrıca, araçlar (eldiven giymek) çok soğuk olduğu önemlidir; Oaletleri ile dokunulduğunda therwise, doku kısmen çözülme olabilir. Bu adımların hepsi AFS odasının içinde yapılır ve cihazlar odasının kenarına üzerinde hareket etmek asla izin verilmez. Benzer şekilde, AFS kullanılan bütün kimyasal maddelerin uygun soğutma gereklidir. <ol><li> -Dalma donma bir EM kriyoprezervasyon enstrüman (TBM) içinde -184 ° C&#39;de sıvı propan retina şeritler. ince bir fırça kullanarak, (fırça kullanarak ekstra tampon kapalı fitil) dalan çubukların ucunda gitmek metal taslakları bağlı çift sopa bant küçük parçalar üzerinde örnekleri yerleştirin. </li><li> Sıvı propan içine çubuklar dalma ve yaklaşık 5 saniye boyunca orada tutun ve sonra sıvı nitrojen ile doldurulmuş küçük bir taşıma odasını kullanarak otomatik dondurarak ikame enstrüman (AFS) transfer ( &#39;LN 2 cryo transferi konteyner soğutmalı&#39;). </li><li> AFS içine donmuş örnek ve aletleri (forseps ve neşter) yerleştirdikten sonra, se için odasında aletleri serinVeral dakika doku dokunmadan, ya da küçük nakil odasına birkaç saniye için araçların ipuçları koyarak bunları soğutmak önce (sıvı azot ile dolu () &#39;LN 2 cryo transferi konteyner soğutmalı&#39;). </li><li> AFS kez ince bir neşter kullanılarak saplama numune ve bandı çıkarın. Ayrıca, azot gazı akış kontrol altında tutmak, TF bu işlemler sırasında açık (TF &#39;LN 2 buharlaştırıcı için regülatör kontrol&#39; olarak tarif edilmiştir). </li><li> (AFS kurmadan önce, yani) düz gömme sahipleri içine donmuş doku yerleştirmeden önce, şeffaf bir plastik levha ince bir daire kesip ve her iyi alt hattına kadar tutucu dibine yerleştirin. Bittiğinde Bu polimerize örnek blok görece daha kolay çıkarılmasını sağlar. NOT: Daha önce, çift Reichert + jelatin kapsülleri (Petralia ve Wenthold, 1999 bakınız) istihdam düz katıştırma sahiplerine alternatif bir yöntem kullanılabilir, ve &lt;sup&gt; 20. </li><li> = 4 ° C / saat (11.3 saat), T2 ile 32 saat, S1 = artış sıcaklığı için T1 = -90 ° C -45 ° C 50 saat, S2 için: AFS (enstrüman terminolojiyi kullanarak) aşağıdaki otomatik dizisini kullanın = 5 ° C / saat (9 saat), (toplam = 142.3 saat) 40 saat süre ile T3 = 0 ° C artış sıcaklığı. önce zamanlı dizisi başlamadan (-90 ° C&#39;de) AFS örnekleri yerleştirmek için 4 saat - 2 adet sürebilir. </li><li> AFS&gt; 32 saat boyunca -90 ° C&#39;de metanol içinde% 1.5 uranil asetat içinde dondurulmuş bölümler bırakın. Düz gömme alüminyum tutucu yedi kuyu her dokuların (tipik olarak) iki adet yerleştirin (üç AFS uyması). <ol><li> kuyularda uranil asetat / metanol içine doku + bandı yerleştirin ve bant doku kaldırmak çok zor ise, hemen önce infiltrasyon (örneğin Lowicryl HM20 gibi) başlangıç ​​gömme ortamına, daha sonra bandı çıkarın. </li></ol></li><li> Daha sonra sıcaklık adım adım artış, saat başına -45 ° C (+ 4 ° C; Otomatik sırayla). </li><li> Her düz gömme sahibinden eski çözüm kaldırmak için ince uçlu bir plastik pipet kullanarak, ve sonra soğutulmuş taze metanol eklemek için başka bir standart plastik pipet kullanarak, önceden soğutulmuş metanol numuneleri üç kez yıkayın. </li><li> 1 ve 2: Daha sonra, aynı yöntemi kullanarak, örneğin 1 de ortamı (HM20 / metanol gömme kütlesi olarak düşük sıcaklık gömme reçine ile kademeli örnekleri infiltre 2 saat boyunca saf reçine ardından 2 saat süre ile, 1 her biri, ve daha sonra reçine değiştirme ve) O / N tutun. </li><li> Sadece kuyu üst kenarına ulaşmak için reçine seviyesinin ayarlanması, ertesi gün tekrar reçine değiştirin. </li><li> örnekleri polimerize (-45 ° C, otomatik sırayla 0 ° C&#39;ye kadar, saatte ortalama + 5 ° C) 40 saat süre ile, UV ışığı ile. </li><li> Sonra AFS örnekler kaldırın. Tipik olarak, örnek bloklar hala renkli bazı pinkness göstermektedir. RT O kimyasal davlumbaz floresan ışık yakın örnekleri, koyun / N veya daha uzun onlar kadar uygulamaKulak tamamen net. </li></ol> 3. Postembedding EM immunogold İmmünositokimya Not: 23,24,25 tarif edildiği gibi Postembedding immünositokimya gerçekleştirilir. <ol><li> % 1 toluidin mavisi ile 1 mikron Ultramikrotomla bölümleri, leke bölümleri kesmek ve bölüm oryantasyonu için onları incelemek; Kesit optimal enine uçağı elde etmek için doku bloğu yönlendirmek. </li><li> Ultramikrotomla 70 nm kalınlığında ultra ince bölümleri kesmek ve Formvar-karbon kaplı nikel yuvası ızgaraları onları toplamak. </li><li> Yıkama bir Tris-tamponlu tuzlu su, üç kez (TBS, 0.05 M Tris tamponu,% 0.7 NaCl, pH 7.6) yıkama izledi damıtılmış H2O içinde bir kez ağlar. </li><li> ve bir NMDAR alt-birimi (anti-tavşan GluN2A 1:50 ila bir antikor: keçi anti-CTB ihtiva eden bir karışım 20 ul damla (3,000 1) daha sonra, 30 dakika TBS içinde% 5 BSA, 20 ul damlalar ızgaraları inkübe ve , GluN2B 1:30), ya da bir anti-tavşan GluA2 / 3 (1:30)% 2 BSA ile TBS-Triton (TBST,% 0.01 Triton X-100, pH 7.6) ve 0.02 M Nan 3 O / RT N. </li><li> 5 dakika TBS (pH 8.2) ve ardından üç ayrı 10 için TBS damla (20 ul) (pH 7.6), 10, ve 20 dk yıkama ızgaraları. </li><li> TBST içinde 18 nM altın parçacıklarına bağlanmıştır 10 nm altın parçacıkları ve eşek anti-keçi IgG (1:20) bağlı eşek anti-tavşan IgG (1:20) ihtiva eden bir karışım damla 2 saat (20 ul) için ızgaraları inkübe 2% BSA ve 0.02 M NaN 3 (pH 8.2). </li><li> ultra saf su içinde, daha sonra 5, 5 ve 10 dakika süre ile TBS 20 ul damlalar (pH 7.6) &#39;de ızgaraları yıkayın ve kurutun. </li><li> Sırasıyla, karanlık koşullarda 8 ve 5 dakika için damıtılmış H2O içinde% 5 uranil asetat ve% 0.3 kurşun sitrat ile karşı-ızgaralar. </li><li> Paket etiketleme deneyleri için, ızgaralar inkübe O / anti-keçi CTB bir karışımının 20 ul damla (1: 3000) oda ısısında N, anti-fare PSD-95 (1: 100) ve anti-tavşan GluN2A (1 : 50). Ardından 2 2 saat süreyle ızgaraları inkübe2% BSA ve 0.02 M NaN 3 TBST içinde 18, 10, ve 5 nm ve altın parçacıklarına bağlanmıştır IgG&#39;lerin bir karışımı (pH 8.2) içinde 0 ul düşer. Yıkama arasında ve antikor inkübasyondan sonra counterstaining çift etiketleme gibi aynı prosedürleri tutun. </li><li> Kontroller ikincil antikorlar, tek başına uygulandığında edildiği yapılmıştır. </li><li> Bir EM isimli ızgaraları ve digitalize görüntüler. 24 gerekirse, sadece parlaklık ve kontrast için Adobe Photoshop 6.0 ​​final rakamları işleyin. </li></ol> 4. sayısallaştırılması <ol><li> El ile 8,000X büyütmede delik veya çatlak olmadan iç pleksiform tabakasının (IPL) seçkin alanlar, daha sonra rastgele 25,000X büyütmede IPL tam derinliğini fotomontaj. </li><li> bunlar: a) retrograd taşınan CTB sinyali (büyük altın parçacıkları) içerir, b) sergiler iyi tanımlanmış membranlar, yarıklar ve postsinaptik yoğunlukları, c) en az iki küçük altın bölümü içerdiğinde konik bipolar çiftlerinde de RGC dendrit tespitPSD içindeki icles veya extrasynaptic plazma zarı boyunca, birden fazla küçük bir altın parçacığı. </li><li> kantitatif analiz için yukarıdaki kriterlere göre, 55 RGC dendrit - 45 toplayın. </li><li> PSD ve NIH ImageJ yazılımı ile bireysel RGC dendritler extrasynaptic plazma zarı uzunlukları ölçün. Membranın 10 nm olan altın parçacıkları sayısı 7, bir ortalama kalınlığına göre, membran bağlantılı - plazma zarı 26 9 nm. </li><li> Doğrusal mikrometre (altın / um) başına altın parçacıkları sayısı parçacık yoğunluğu hesaplayın. Her altın parçacığının merkezi ve (sinaptik konumu için) PSD ortasında ya da (extrasynaptic konumu için) PSD kenarı arasındaki mesafeyi ölçün. </li><li> yazılım tarafından istatistiksel analiz. karşılaştırmak için iki kuyruklu t-testi kullanın. önemi p &lt;0.05 varıldı. Aksi belirtilmedikçe, rapor değerleri ± SEM 18 demek. </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabotropic+glutamate+receptor+(mGluR1)+is+concentrated+at+perisynaptic+membrane+of+neuronal+subpopulations+as+detected+by+immunogold+reaction.">The metabotropic glutamate receptor (mGluR1) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.</a> Neuron. 11, 771-787 (1993).</li><li>Van Lookeren Campagne, M., Oestreicher, A. B., van der Krift, T. P., Gispen, W. H., Verkleij, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-substitution+and+Lowicryl+HM20+embedding+of+fixed+rat+brain:+Suitability+for+immunogold+ultrastructural+localization+of+neural+antigens.">Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: Suitability for immunogold ultrastructural localization of neural antigens.</a> J. Hist. Cyt. 39, 1267-1279 (1991).</li><li>Matsubara, A., Laake, J. H., Davanger, S., Usami, S., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+AMPA+receptor+subunits+at+a+glutamate+synapse:+A+quantitative+immunogold+analysis+of+hair+cell+synapses+in+the+rat+organ+of+Corti.">Organization of AMPA receptor subunits at a glutamate synapse: A quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.</a> J. Neurosci. 16, 4457-4467 (1996).</li><li>Petralia, R. S., 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=Organization+of+NMDA+receptors+at+extrasynaptic+locations.">Organization of NMDA receptors at extrasynaptic locations.</a> Neuroscience. 167, 68-87 (2010).</li><li>Merighi, A., Polak, J. M., Priestley, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embedding+electron+microscopic+immunocytochemistry.">Post-embedding electron microscopic immunocytochemistry.</a> Electron Microscopic Immunocytochemistry: Principles and Practice. , 51-87 (1992).</li><li>Ottersen, O. P., Takumi, Y., Matsubara, A., Landsend, A. S., Laake, J. H., Usami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+organization+of+a+type+of+peripheral+glutamate+synapse:+the+afferent+synapses+of+hair+cells+in+the+inner+ear.">Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear.</a> Prog. Neurobiol. 54, 127-148 (1998).</li><li>Nusser, Z., Lujan, R., Laube, G., Roberts, J. D., Molnar, E., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+and+pathway+dependence+of+synaptic+AMPA+receptor+number+and+variability+in+the+hippocampus.">Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.</a> Neuron. 21, 545-559 (1998).</li><li>Takumi, Y., Ramirez-Leon, V., Laake, P., Rinvik, E., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+modes+of+expression+of+AMPA+and+NMDA+receptors+in+hippocampal+synapses.">Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.</a> Nat. Neurosci. 2, 618-624 (1999).</li><li>Grimes, W. 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=Complex+inhibitory+microcircuitry+regulates+retinal+signaling+near+visual+threshold.">Complex inhibitory microcircuitry regulates retinal signaling near visual threshold.</a> J. Neurophysiol. 114, 341-353 (2015).</li><li>Sassoe-Pognetto, M., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+ionotropic+glutamate+receptors+at+dendrodendritic+synapses+in+the+rat+olfactory+bulb.">Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb.</a> J. Neurosci. 20, 2192-2201 (2000).</li></ol>

The authors have no disclosures.

Başarılı kantitatif sonrası gömme immunogold EM için dört teknikleri tarif etmişlerdir: 1) kısa ve zayıf tespit, 2) donma-ikamesi, 3)-sonrası gömme immunogold boyama ve 4) ölçümü. EM immünolojik ultra ince doku kesitlerinde spesifik proteinlerin belirlenmesini sağlar. altın partikülleri ile etiketlenmiş antikorlar ile doğrudan EM kullanılarak görüntülenebilir. Bir zar reseptörü subsynaptic lokalizasyonu tespit güçlü iken, EM immünolojik teknik açıdan zor ola...

Glutamat retinada 1 majör eksitatör nörotransmiter olduğunu. Bipolar hücreleri 2 glutamaterjik sinaptik girdi alan retina ganglion hücrelerinin (RGCs), beyne görsel bilgi göndermek retinanın çıkış nöronları vardır. Fizyolojik çalışmaları RGCs sinaptik uyarım NMDA reseptörlerinin (NMDARs) ve AMPA reseptörleri (AMPARs) 3,4,5 tarafından postsynaptically aracılık ettiğini göstermiştir. RGCs eksitatuvar postsinaptik akımlar (EPSCs) AMPARs ve NMDARs aracılık ettiği, ancak RGCs ilgili 3,5,6,7,8 spontan minyatür EPSCs (mEPSCs) sadece AMPARs aracılı bileşen 4,5,9 sergiler. Ancak, glutamat alımını azaltarak RGC dendritler üzerinde NMDARs uyarıcı sinaps dışında olabilir düşündüren, spontan EPSCs 5 bir NMDAR bileşeni saptandı. Membran ilişkili guanilat kinazlar PSD-95 olarak (MAGUKs) o glutamat reseptörleri ve iyon kanalının da dahil olmak üzere küme nörotransmitter reseptörleri,sinaptik s, ayrıca ayrı subsynaptic ekspresyonu 10,11,12,13,14 sergiler. Son yıllarda, konfokal immünohistokimya ve ön gömme elektron mikroskobu (EM) immünohistokimya üzerinde zar reseptör ekspresyonunu incelemek için istihdam edilmiştir. konfokal immün reseptör ekspresyonunun geniş desenler ortaya koymaktadır rağmen, onun düşük çözünürlüklü imkansız hücre içi konumu ayırt etmek kullanmayı kolaylaştırır. Memeli retinada ön gömme EM çalışmaları NMDAR alt birimleri koni bipolar hücre şerit sinaps 15,16,17 de postsinaptik elemanları mevcut olduğunu göstermektedir. Bunun sebebi, fizyolojiksel kanıt açıkça aksine. Bununla birlikte, reaksiyon ürünü difüzyon ön gömme immünoperoksidaz yöntemi iyi bilinen bir sonucudur. Bu nedenle, bu yaklaşım genellikle istatistiksel güvenilir veriler vermez ve extrasynaptic membran 18,19,20,21 karşı sinaptik membran lokalizasyonu arasındaki ayrımı hariç tutabilir. üzerindeÖte yandan, fizyolojik ve anatomik veriler RGCs 3,5,7,9,22 üzerine AMPARs bir sinaptik lokalizasyonu ile uyumludur. Böylece, retina şerit sinaps glutamat reseptörleri ve MAGUKs postsinaptik değil, aynı zamanda perisynaptic veya extrasynaptic zar bölmelerine sadece lokalizedir. Ancak, retina şerit sinaps bu zar proteinlerinin yüksek çözünürlüklü kantitatif analiz hala ihtiyaç duyulmaktadır. Burada, kolera toksin altbirim B kullanılarak etiketli (CTB) sıçan RGCs üzerine sinaps bu proteinlerin sayısı, yoğunluğu ve değişkenliği tahmin izledi NMDAR alt birimden, Ampar alt birimlerin ve PSD-95 subsynaptic lokalizasyonu incelemek için bir postembedding EM immunogold tekniği geliştirdi retrograd izleme yöntemleri.

<table><tbody><tr><td>Paraformaldehyde</td><td>EMS</td><td>15710</td><td></td></tr><tr><td>Glutaraldehyde</td><td>EMS</td><td>16019</td><td></td></tr><tr><td>NaH2PO4</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Na2HPO4</td><td>Sigma</td><td>7782-85-6</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C-8106</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A-7030</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>221465</td><td></td></tr><tr><td>NaN3</td><td>JT Baker</td><td>V015-05</td><td></td></tr><tr><td>Glycerol</td><td>Gibco BRL</td><td>15514-011</td><td></td></tr><tr><td>Lowicryl HM 20</td><td>Polysciences</td><td>15924-1</td><td></td></tr><tr><td>Tris-Base</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>04997-100</td><td></td></tr><tr><td>Anti-GluN2A</td><td>Millipore</td><td>AB1555P</td><td>Dilution 1/50</td></tr><tr><td>Anti-GluN2B</td><td>Millipore</td><td>AB1557P</td><td>Dilution 1/30</td></tr><tr><td>Anti-GluA2/3</td><td>Millipore</td><td>AB1506</td><td>Dilution 1/30</td></tr><tr><td>Anti-PSD-95</td><td>Millipore</td><td>MA1&amp;ndash;046</td><td>Dilution 1/100</td></tr><tr><td>Donkey anti-rabbit IgG-10 nm gold particles</td><td>EMS</td><td>25704</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-10 nm gold particles</td><td>EMS</td><td>25814</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-5 nm gold particles</td><td>EMS</td><td>25812</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-goat IgG-18 nm gold particles</td><td>Jackson ImmunoResearch</td><td>705-215-147</td><td>Dilution 1/20</td></tr><tr><td>Formvar-Carbon coated nickel-slot grids.</td><td>EMS</td><td>FCF2010-Ni</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400-1</td><td></td></tr><tr><td>Methanol</td><td>EMS</td><td>67-56-1</td><td></td></tr><tr><td>Lead citrate</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM AFS</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM CPC</td><td>Leica</td><td></td><td></td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td></td><td></td></tr><tr><td>JEOL 1200 EM</td><td>JEOL</td><td></td><td></td></tr><tr><td>liquid nitrogen&amp;nbsp;</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>Propane</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>CTB</td><td>List Biological Laboratories</td><td>104</td><td>1 - 1.2%</td></tr><tr><td>Anti-CTB</td><td>List Biological Laboratories</td><td>703</td><td>Dilution 1/4,000</td></tr></tbody></table>

high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

Daha önce 24,25 açıklandığı gibi burada sunulan sonuçlar, sıçan retina RGC dendritler üzerinde çarpıcı farklı subsynaptic lokalizasyon GluA 2/3 kalıplarını ve NMDARs göstermektedir. RGC dendritik profillerde GluA 2/3 immünolojik parçacıklarının% 77, merkezi sinapslara benzer PSD (Şekil 1A) içinde yer. Ancak, NMDARs ya sinaptik veya extrasynaptically bulunurdu. GluN2A immünolojik partiküllerin% 83 PSD lokalize edilmiştir (Şe...

Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retina Şerit Sinapslar at Membran Reseptör Yüksek Çözünürlüklü Sayısal immunogold Analizi

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

high-resolution-quantitative-immunogold-analysis-membrane-receptors

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Neuroscience

7.5K Görüntüleme.  National Institutes of Health. The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Video: High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Sinapslar miktarının: Synapse Numarası ölçmek için bir İmmünositokimya tabanlı Testi

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Nörobilim

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Retina Bipolar Hücreler TIRF Mikroskopi Görüntüleme ekzositoz

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Biyoloji

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Hipokampal Dilim Kültürleri Synaptic Proteinler Post-gömme immunogold Etiketleme

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Wholemount Sıçan Retina Immunohistochemical ve Kalsiyum Görüntüleme Yöntemleri

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Optogenetic ve donma-fraktür çoğaltma ile immün Fare amigdalada glutamat reseptörlerinin giriş-özel bir düzenleme incelemek için bir araya

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

İnsan İmmünolojik Sinaps Görüntüleme

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

İmmünoloji ve Enfeksiyon

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Görüntüleme ve İmmünhistokimyanın için Retina Wholemount Hazırlık için optimize edilmiş Protokolü

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

<meta charset="utf8"></head><body>TEM için Retinal Organoid Örnek Hazırlama için Optimize Edilmiş Protokol

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

<meta charset="utf8"></head><body>Yazar Spotlight: TEM Kullanarak Olgun Retinal Organoidlerde Sinaptik Ultrastructure'ın Analiz Edilmesi

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Transmisyon elektron mikroskobu için retinal organoid örneklerinin hazırlanması

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

Retina Şerit Sinapslar at Membran Reseptör Yüksek Çözünürlüklü Sayısal immunogold Analizi

Özet

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İnsan İmmünolojik Sinaps Görüntüleme

Görüntüleme ve İmmünhistokimyanın için Retina Wholemount Hazırlık için optimize edilmiş Protokolü

Transmisyon elektron mikroskobu için retinal organoid örneklerinin hazırlanması

Transmisyon elektron mikroskobu için retinal organoid örneklerinin hazırlanması

Transmisyon elektron mikroskobu için retinal organoid örneklerinin hazırlanması