Yazarlar, laboratuvar uzmanlık için reçine gömme ve Bayan Stephania Mezarları Bayan Sue Connell onun uzmanlık yardımı kabul etmek istiyorum. Yazarlar, onların tavsiyelerini Profesör Frank Kandziora ve Dr Marie-Anne Polboth teşekkür etmek istiyorum.

<ol><li> Dokusu,% 10 fosfat içeren kapalı bir kapta tamponlu formalin 3 içinde yer alır . Florokrom analizi fluorochromes ışığa duyarlı olarak, olması ise, minimal ışığa maruz kalma olun. </li><li> Doku hangi yönlendirme gerekli olacak kurulması. </li><li> Numune boyutuna kesin, doğru yönde, bir grup gördüm. Emin olun güvenlik önlemleri yapıştırılır. Numune sonra opak bir kap içine konur, hacmi yeterli fiksasyon elde etmek için yaklaşık on katı numune boyutu olmalıdır. Numune boyutuna bağlı olarak bir ila iki hafta olmak üzere toplam formalin kalmalıdır. Bu kontrolü doku testi ve optimizasyonu amaçlar için kullanılmasını önerdi. </li><li> Doku gömme kalıp sığar doku ve% 70 etanol içeren, kapalı bir opak kap içine yerleştirin. </li><li> Işık ve sürekli ajitasyon altında koruyucu, etanol konsantrasyonları artan doku işleyin. <ol class="lalpha"><li> % 70 etanol 1 hafta </li><li> % 80 etanol 1 hafta </li><li> % 90 etanol 1 hafta </li><li> % 100 etanol 1 hafta </li></ol></li><li> Daha sonra doku, ışık ve sabit ajitasyon altında tekrar koruyucu 1 hafta bütanol temizlenir. </li><li> Reçine ve kemik yoğunluğu yakından reçine yoğunluğu (bkz. adım 8) değiştirilebilir eşlemeli ve doğru yoğunluğu sağlamak için ilk olarak bir test örneğinin gömme tavsiye edilmelidir. Biz ışık mikroskobu ve florokrom analiz için Technovit 7100 (Heraeus Kulzer) kullanın. Immünohistokimya gerekiyorsa alternatif bir reçine gibi Technovit 9100 (Heraeus Kulzer) olarak kullanılması gerekir. </li><li> Technovit 7100 hazırlık çözüm gerekirse, kit ile birlikte verilen yumuşatıcı çözüm küçük alikot ile karışık olabilir, ancak en fazla 5% hazırlık çözüm hacim; deneyimlerimiz yumuşama çözüm nadiren gereklidir. Mutlak etanol ve baz sıvı Technovit 7100 eşit parçaya 24 saat ışığa maruz kalma en aza indirmek için bir ön infiltrasyon çözüm olarak ve ajitasyon ile karıştırılır ve doku uygulanır. </li><li> Infiltrasyon çözüm sonra, 100 ml tabanı sıvı içinde çözünmüş 1 gr sertleştirici I (= 1 torba) kullanılarak yapılır. Bu ön infiltrasyon çözüm yerine sürekli ajitasyon altında 1 hafta sızmak için izin verilir. </li><li> Aşağı kesim yüzeyi yüz doku, kalıp içine yerleştirin. (Ideal olarak en az 5 milimetre) reçine için doku etrafında bir marj var olduğundan emin olun; bu blok gücü sağlar. 1 sertleştirici II ml Technovit 7100 hazırlık çözüm 15mls karıştırın ve kalıp numune etrafında dökün ve en az 5 millimietres kapağı. Kür 2 saat içinde başlayacaktır, ancak bir gecede tamamlanmış olacaktır. </li><li> Montaj Technovit 3040 (Heraeus Kulzer) kullanarak bir yedekleme blok fashioning sonra yapılır. Toz 1 kısım sıvı viskoz bir sıvı elde etmek için 2 parça bir hacim oranı 3040 Technovit karıştırın. Technovit 3040 blok blok temel üzerinde en az 2 mm bir seviyeye geri girinti içine dökün. Yaklaşık 10 dakika sonra kalıp uzak soyulmuş ve blok hazır olabilir. </li><li> Yoğunluğu ve bu nedenle metakrilat reçine kesme özellikleri, odadaki ortam nem duyarlı. Bloklar dolayısıyla desikatöre saklanmalıdır. </li><li> Zemin kesit kalınlığı 20-50 mikrometre arasında büyük bölümleri üretir. Kalın bölümlerde parlak floresan üreten (bölümüne bakınız) florokrom analizi için yararlı olur. Macrotome elmas bıçak sabitleyin. Macrotome rezervuar yağ, petrol ruhu gibi ekleyin. Güvenli ve kelepçe blok bıçak yolunuzu. İzin yeterli yağlama sağlar (Bölüm başına yaklaşık 20-30 dakika) yavaş yavaş ortaya taşlama. Ilk bölümü kesme yüzünü ortaya çıkarır ve bölümü atılmalıdır yönlendirir. </li><li> Bir sonraki bölümde, ardından bir SuperFrost Plus slayt yerleştirilir. Bu bölüm, bunun üzerinde bir sandviç torbası bir kısmını yerleştirerek ve daha sonra başka bir slayt yerde kelepçeli düz tutarak tavsiye ederiz kıvrılabilecek. Daha sonra 1 saat boyunca sıcaklık 60-80 derece arasında bir inkübatör içine yerleştirilir. Bu metakrilat yumuşatır ve düz bir şekilde slayt bölümünde uymak yardımcı olur. </li><li> Nazik bir şekilde ince kum zımpara kullanarak gerekli zemin bölümünde temizlenir ve parlatılır olabilir. </li><li> Kızak mikrotom, ince kesitler keser ışık microsocopy için en iyi bölümleri sağlar. Bu sabit bloklar kesim için gerekli sertliği, gibi bir kızak mikrotom kullanın. Alternatif olarak çalışan döner mikrotom kullanılabilir. Keskin bir paslanmaz çelik bıçak kullanın. Bu bir kullanılan diğer olarak bilenmiş olarak kullanılabilir iki ağza sahip olmak tercih edilir. Bıçak bloğu ile 45 derecelik bir açı yapmalıdır. Surface yumuşatma gerekirse engellemek için ıslak bir kağıt uygulayarak elde edilebilir. Bu kaliteli bir bölümünü elde etmek için istenilen kesme koşulları elde etmek için çeşitli bölümler sürebilir. Bu bölüm, daha sonra bir su banyosu üzerinde yüzen ve 16 olduğu gibi bir slayt yerleştirilir. </li><li> Boyama reaktifler, aşağıda Malzeme listelenmiştir. Protokoller, Bancroft 4 ve ark . Kalınlık değişimi nedeniyle test slaytlar üzerinde lekeler optimize etmek için tavsiye edilir. Raf boyama doku gerekebilir doğrudan düz bir slayt lekeleri ekleyerek böylece slayt float neden olabilir. Özdeş bir protokol ya da raf boyama histomorfometri için gerekli tutarlı boyama sonuçlar için gereklidir. <ol class="lalpha"><li> Hematoksilen ve Eosin <ol class="lroman"><li> Haematoxyilin 5-10 dakika ile Leke </li><li> Scott, musluk suyu ile yıkayın </li><li> Eozin 5 dakika ile Leke </li><li> Musluk suyunda yıkayın </li><li> Ksilen içinde Temizle </li><li> Dağı </li><li> Sonuçlar <ol><li> Osteoid = Pembe </li><li> Kalsifiye kemik = morumsu kahverengi </li><li> Çekirdekler = mavi </li></ol></li></ol></li><li> Von Kossa <ol class="lroman"><li> Gümüş nitrat çözeltisi koyun ve güçlü ışığa maruz mineralize kemik siyah olana kadar (yaklaşık 10 dakika) </li><li> Üç kez distile su ile yıkayın </li><li> 5 dakika boyunca sodyum tiyosülfat ile Tehdit </li><li> Distile su ile yıkayın </li><li> Safrinin O ile Counterstain </li><li> Ksilen içinde Temizle </li><li> Dağı </li><li> Sonuçlar <ol><li> Mineralised kemik = black </li><li> Osteoid = kırmızı / pembe </li></ol></li></ol></li><li> Masson Goldner s trikrom <ol class="lroman"><li> (% 80 etanol 90mls ve 20 dakika boyunca% 25 oranında amonyak 10mls) alkalin alkol ile yıkayın. </li><li> Suyla yıkayın </li><li> Distile su ile durulayın </li><li> 10 dakika boyunca Weigert s Hematoksilen ile Leke </li><li> Distile su ile durulayın </li><li> Ponceau-Fuchsin nihai çözüm 5 dakika ile Leke </li><li> % 1 asetik asit ile 15 saniye çalkalayın </li><li> Phosphomolybdic asit-turuncu G çözüm 5 dakika içinde Leke </li><li> % 1 asetik asit ile 15 saniye çalkalayın </li><li> 5 dakika açık yeşil ile Leke </li><li> % 1 asetik asit ile durulayın 3 değişiklik </li><li> Distile su ile durulayın </li><li> Dağı </li><li> Sonuçlar <ol><li> Mineralised kemik = yeşil </li><li> Osteoid = kırmızı / turuncu </li><li> Çekirdekler = mavi gri </li><li> Kıkırdak = mor </li></ol></li></ol></li></ol></li><li> Florokrom hazırlıkları: </li><li> Bunlar en az iki hafta arayla, yavaş intravenöz enjeksiyonu ile idare edilmektedir. </li><li> Dozlar <ol class="lalpha"><li> Calcein Yeşil 10 mg / kg IV </li><li> Oksitetrasiklin 50 mg / kg IV </li><li> Alizarin Complexone 30 mg / kg IV </li></ol></li><li> Calcein Yeşil hazırlanması <ol class="lalpha"><li> Calcein Yeşil Bir gram eriyene kadar 1 M NaOH ile titre edilir. </li><li> pH pH = 7,1-7,2 kadar% 1 NaOH ile ayarlanır. </li><li> Filtre ve ışıktan korumalı tutmak </li></ol></li><li> Alizarin Complexone hazırlanması <ol class="lalpha"><li> Üç gram Alizarin complexone eriyene kadar 1 M NaOH ile titre edilir. </li><li> pH pH = 7,1-7,2 kadar% 1 HCl veya NaOH ile ayarlanır. </li><li> Filtre ve ışıktan korumalı tutmak </li></ol></li><li> Oksitetrasiklin, hazırlanması <ol class="lalpha"><li> Oksitetrasiklin raftan bir anti-mikrobik ilaç olarak kullanılabilir. Herhangi bir hazırlama gerekli değildir. </li></ol></li></ol>

<ol>
	<li>Kerr, 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> Atlas of Functional Histology. , 164-168 (2000).</li><li>Parfitt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+histomorphometry:+standardization+of+nomenclature,+symbols,+and+units.+Report+of+the+ASBMR+Histomorphometry+Nomenclature+Committee.">Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee.</a> J Bone Miner Res. 2, 595-610 (1987).</li><li>An, Y. H., Martin, 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=.">.</a> Handbook of histology methods for bone and cartilage. , (2003).</li><li>Bancroft, J. D., Gamble, 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> Theory and Practice of Histological Techniques. , 352-360 (2008).</li></ol>

Çözücüler: <ul><li> Normal% 10 Formalin, Etanol, ksilen ve Butanol asit Tamponlu Tüm histoloji notu, kükürt ücretsiz. Grale Bilimsel Sanayi ve Ticaret Ltd. http://www.grale.com.au </li></ul> Kalıplar: <ul><li> Uzakta Peel, plastik kalıpları Grale Bilimsel </li></ul> Slaytlar: <ul><li> SuperFrost Plus Mikroskop slaytlar Lomb Bilimsel Sanayi ve Ticaret Ltd. <a href="http://www.lomb.com.au/">http://www.lomb.com.au</a> </li></ul> Reçine: <ul><li> GlycolMethacrylate Technovit 7100 Heraeus Kulzer <a href="http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm">http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm</a> </li><li> Blok Reçine-Technovit 3040 Histolojik blok, bir Heraeus Kulzer </li></ul> Lekeler (Grale Bilimsel Reaktifler) Goldner Kullanıcı trikrom leke </li> 1. Weigert demir hemotoxylin (DeGroat) <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a) Hemotoxylin çözüm </td></tr><tr><td> Hemotoxylin </td><td> 1g </td></tr><tr><td> Saf alkol </td><td> 100 mL </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) Demir Çözüm </td></tr><tr><td> % 30 sulu demir klorür </td><td> 4ml </td></tr><tr><td> Hidroklorik asit (konsantre) </td><td> 1mL </td></tr><tr><td> distile su </td><td> 95mL </td></tr></table> Filtrelenmiş ve eşit hemotoxylin hacmi hemen önce leke kullanılan eklendi. 2. Ponceau-fuksin-azophloxin stok çözümleri <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a) Ponceau de xylidine çözüm </td></tr><tr><td> Ponceau de xylidine </td><td> 0.75g </td></tr><tr><td> asit fuchsion </td><td> 0.25g </td></tr><tr><td> Asetik asit </td><td> 1mL </td></tr><tr><td colspan="2"> mix ve distile su (100 ml) </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) azophloxin çözüm </td></tr><tr><td> Azophloxin </td><td> 0.5g </td></tr><tr><td> Asetik asit </td><td> 0.6mL </td></tr><tr><td colspan="2"> mix ve distile su (100 ml) </td></tr><tr><td colspan="2" style="font-weight:bold;"> c) Final çalışma leke çözüm </td></tr><tr><td> Ponceau-fuksin çözüm </td><td> 5-10ml </td></tr><tr><td> Azophloxin </td><td> 2mL </td></tr><tr><td> % 0.2 asetik asit çözeltisi </td><td> 88mL </td></tr></table> 3. Açık Yeşil Çözüm <table style="margin-left:40px;" border="0"><tr><td> Açık Yeşil </td><td> 1g </td></tr><tr><td> asetik asit </td><td> 1mL </td></tr><tr><td colspan="2"> Mix ve distile su (500ml) </td></tr></table> 4. Phophomolybdic asit-Turuncu G çözüm <table style="margin-left:40px;" border="0"><tr><td> Phosphomolybdic asit </td><td> 3g </td></tr><tr><td> Turuncu G </td><td> 2g </td></tr><tr><td colspan="2"> 500ml distile su içinde çözülür ve kristal timol ekleyin. </td></tr></table> Von Kossa KORUYUCU <table style="margin-left:40px;" border="0"><tr><td> % 1 sulu gümüş nitrat </td></tr><tr><td> % 2,5 sodyum tiyosülfat </td></tr><tr><td> % 1 safranin O </td></tr></table> Alsiyan mavi KORUYUCU <table style="margin-left:40px;" border="0"><tr><td> Alsiyan mavi 8GX </td><td> 1g </td></tr><tr><td> % 3 asetik asit çözeltisi </td><td> 100 mL </td></tr></table> Hemotoxylin ve Eozin leke <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a) Harris hemotoxylin </td></tr><tr><td> Hemotoxylin </td><td> 2.5g </td></tr><tr><td> Saf alkol </td><td> 25ml </td></tr><tr><td> Potasyum Şap </td><td> 50g </td></tr><tr><td> distile su </td><td> 500ml </td></tr><tr><td> cıva oksit </td><td> 1.25g VEYA </td></tr><tr><td> Sodyum iyodat </td><td> 0.5g </td></tr><tr><td> Asetik sirke asidi </td><td> 20ml </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) Eozin çözüm </td></tr><tr><td colspan="2"> Ticari olarak üretilen Eozin kullanılmıştır. </td></tr></table> Tüm leke boya tozları Gurr boya tozları. Bütün çözeltiler analitik reaktifler ve Laboratuar reaktifleri Fluorochromes <table border="1" cellspacing="0" cellpadding="0"><tr><td> Üretici </td><td> Florokrom </td><td> Katalog Numarası </td></tr><tr><td> Sigma-Aldrich, Sydney </td><td> Calcein Yeşil </td><td> C0875 </td></tr><tr><td> Sigma-Aldrich, Sydney </td><td> Alizarin Complexone </td><td> A3882 </td></tr><tr><td> Intervet Avustralya, Victoria </td><td> Oksitetrasiklin (Raf antimikrobiyal ilaç kapalı) </td><td> Unvanı; Engemycin 100 </td></tr></table>

undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis

Undecalcified kemik histolojisi kemik mikro-mimari göstermektedir. Bu mineralize kemik, kemik döngüsü ve kemik yapım ve yıkım ile ilgili önemli bilgiler sağlar ve hücresel bileşenleri gösterir. Bu, klinik ve araştırma uygulamaları çeşitli büyük bir öneme sahiptir. Çok güzel görüntüler 1 verim ve florokrom değerlendirme ve histomorfometri 2 gibi teknikler sağlar. Florokrom analizi, o bir zamanda mineralizasyon miktarı ölçümü için sağlar belirli bir zaman noktasında, kalsiyum bağlamak floresan boya enjekte edilir bir tekniktir. Histomorfometri mikroskobik düzeyde kemik nicelikleme bir süreçtir. Undecalcified kemik histolojisi Sahne teknik özellikle büyük boyutlu örnekleri zordur. Histoloji gömülü standart parafin kullanılan bu tekniği farklılıklar gerektirir. Bu video kaliteli bölümleri üretim sürecini göstermektedir ve bunların üstesinden gelmek için hangi teknik zorluklar ve yöntemler göstermektedir. Genellikle birkaç hafta Ancak, yoğunluk ve düşük kemik geçirgenliği oldukça uzun fiksasyon ve işlem süreleri gereklidir nedeniyle diğer yumuşak dokularda benzer bir şekilde, numune hazırlama, fiksasyon ve işlenmesi ile elde edilir. Gömme metakrilat esaslı reçineler gibi kemik, benzer ya da eşit sertlik ve yoğunluk ile destekleyici bir ortam kullanılarak, ancak elde parafin infiltrasyon ve gömme aksine, bu geri dönüşü olmayan bir bir adımdır. Kesit florokrom analizi gibi çalışmalar için en uygun kalın bir bölümü, üretir öğütülmesi ile elde edilebilir. Bu iyi bir macrotome bir elmas bıçak kullanılarak elde edilir. Alternatif olarak, ince kısımların ışık mikroskobu için üretilen ve bu çok keskin bir bıçak ile bir kızak mikrotom kullanılarak elde. Kızak mikrotom büyük, sert bloklar için gerekli ek güç ve kararlılık sağlar. Reçine gömülü gösterilmektedir lekeleri çeşitli bölümleri ile boyanmış olabilir.

Watch this Scientific Journal Video about Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis at JoVE.com

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified kemik histolojisi, klinik ve araştırma uygulamaların çeşitliliği için önemli bilgiler sağlar. Bu teknik, özellikle büyük boyutlu örnekleri, zordur. Bu video kaliteli bölümleri üretim sürecini göstermektedir ve bunların üstesinden gelmek için hangi teknik zorluklar ve yöntemler göstermektedir.

Histoloji, histomorfometri ve Florokrom Analiz Undecalcified Kemik Hazırlık

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

undecalcified-bone-preparation-for-histology-histomorphometry

Research

JoVE Journal

Biology

27.1K Görüntüleme.  Monash University. Undecalcified kemik histolojisi, klinik ve araştırma uygulamaların çeşitliliği için önemli bilgiler sağlar. Bu teknik, özellikle büyük boyutlu örnekleri, zordur. Bu video kaliteli bölümleri üretim sürecini göstermektedir ve bunların üstesinden gelmek için hangi teknik zorluklar ve yöntemler göstermektedir.

Video: Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Biyoloji

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

Yetişkin hazırlanması Drosophila Gözler İnce Kesit ve Mikroskobik Analizi

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; Yerinde</emTüm Dağı Embriyolar ve Hücre histolojisi&gt; Hibridizasyon Deniz Elasmobranchs İçin Uyarlanmış

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

Kemik koşullu ortam: Hazırlama ve Biyo-

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Kütle Sitometri Analizi için Numune Hazırlama

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM analizi için düşük Hücre sayısı ile örnek hazırlanması için alternatif bir yaklaşım

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Oküler yüzdeki kütle spektrometresi esaslı proteomik analiz için numune hazırlama

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

Synchrotron Micro-CT için Yüksek Verimli Kortikal Kemik Örneği Alımı ve Analizi için Kesitleme, Coring ve Görüntü İşleme Kılavuzu

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

İnsan Osteoartrit Diz Ekleminden Doku Toplama ve RNA Ekstraksiyonu

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...