Os autores gostariam de agradecer o apoio da Sra. Sue Connell por sua especialização na incorporação de resina e Túmulos Ms Stephania para sua perícia em laboratório. Os autores gostariam de agradecer ao professor Frank Kandziora e Dr. Marie-Anne Polboth para os seus conselhos.

<ol><li> O tecido é colocado em um recipiente selado contendo fosfato tamponado a 10% solução de formalina 3. Garantir que a exposição mínima à luz, se a análise fluorocromo é para ser realizado, como a fluorocromos são sensíveis à luz. </li><li> Estabelecer qual a orientação do tecido serão necessários. </li><li> Apare a amostra com o tamanho, na orientação correta, usando uma serra de fita. Medidas de garantir a segurança são cumpridas. A amostra é então colocada em um recipiente opaco, cujo volume deve ser de aproximadamente dez vezes o tamanho da amostra para obter uma fixação adequada. O espécime deve permanecer na solução de formalina um total de entre uma e duas semanas dependendo do seu tamanho. Sugere-se o uso de tecidos de controle para fins de teste e otimização. </li><li> Verifique o tecido se encaixa no molde de incorporação e coloque o tecido em um recipiente selado opaco contendo etanol 70%. </li><li> Processo o tecido em ordem crescente as concentrações de etanol, protegendo da luz e sob agitação constante. <ol class="lalpha"><li> 70% de etanol 1 semana </li><li> 80% de etanol 1 semana </li><li> 90% de etanol 1 semana </li><li> 100% de etanol 1 semana </li></ol></li><li> O tecido é então apagado em butanol para uma semana, mais uma vez blindagem da luz e sob agitação constante. </li><li> A densidade da resina e osso devem ser cuidadosamente acompanhado. A densidade da resina pode ser alterada (ver passo 8) e recomendamos a incorporação de um espécime primeiro teste para garantir a densidade correta. Usamos Technovit 7100 (Heraeus Kulzer) para microscopia de luz e análise fluorocromo. Se imunohistoquímica é necessária uma resina alternativa deve ser usada como Technovit 9100 (Heraeus Kulzer). </li><li> A solução de preparação Technovit 7100 pode ser misturado com pequenas alíquotas da solução de amolecimento fornecido com o kit, se necessário, mas não mais que 5% o volume de solução de preparação, em nossa experiência, a solução de amolecimento raramente é necessário. Partes iguais de etanol absoluto e Technovit base líquida 7100 são misturados e aplicados ao tecido como uma solução de infiltração de pré-por 24 horas minimizando a exposição à luz e com agitação. </li><li> A solução é, então, a infiltração composto usando um endurecedor g I (= saco 1) que é dissolvido em 100 ml de líquido de base. Isto substitui a solução de pré-infiltração e é permitido para se infiltrar em uma semana sob agitação constante. </li><li> Coloque o tecido no molde, com o rosto superfície de corte para baixo. Garantir que há uma margem (de preferência, pelo menos, 5 milímetros) em todo o tecido para a resina, o que garante força bloco. Mix 15mls de Technovit solução de preparação 7100 com 1 ml de endurecedor II e despeje em torno de amostra no molde e cobri-lo em pelo menos 5 millimietres. Cura vai começar dentro de 2 horas, mas será concluída durante a noite. </li><li> Montagem é, então, realizada por formar um bloco de apoio usando Technovit 3040 (Heraeus Kulzer). Mix Technovit 3040 em uma relação de volume de 2 partes de pó para 1 parte de líquido para obter um líquido viscoso. Despeje Technovit 3040 no recesso na parte de trás do bloco a um nível de pelo menos 2 mm acima da base do bloco. Após cerca de 10 minutos, o molde pode ser desfeito, eo bloco está pronto. </li><li> A densidade e, portanto, as propriedades de corte de resina de metacrilato são suscetíveis à umidade ambiente na sala. Os blocos devem ser armazenados em um dessecador. </li><li> Seccionamento terra produz seções maiores entre 20-50 micrômetros de espessura. É útil para a análise fluorocromo (ver secção abaixo) como seções mais espessas produzem mais brilhante fluorescência. Fixe o disco de diamante para macrotome. Adicionar lubrificante, como o espírito de petróleo para o reservatório no macrotome. Garantir o bloco no grampo e orientar a lâmina. Permitem moer a ocorrer lentamente (cerca de 20-30 minutos por seção) garantindo a lubrificação adequada. A primeira seção expõe a face de corte e orienta a seção deve ser descartado. </li><li> A próxima seção é então colocada sobre uma lâmina Além disso Superfrost. A seção tem uma tendência a enrolar por isso, recomendamos colocar uma porção de um saco de sanduíche por cima e, em seguida, segurando-o apartamento com outro slide preso no lugar. É então colocado em uma incubadora de entre 60-80 graus Celsius durante 1 hora. Isto suaviza a metacrilato e ajuda a seção aderir ao deslize de uma forma plana. </li><li> A seção de solo pode ser limpo e polido, conforme necessário usando lixa fina de uma maneira suave. </li><li> O micrótomo sledge cortes cortes finos, que proporciona o melhor seções para microsocopy luz. Use um micrótomo trenó como tem adicionado rigidez, que é necessário para o corte desses blocos rígidos. Alternativamente um micrótomo rotativo movido podem ser usados. Use uma lâmina de aço inoxidável afiada. É preferível ter duas lâminas disponíveis como se pode ser afiada como a outra é utilizada. A lâmina deve fazer um ângulo de 45 graus com o bloco. Suamolecimento rface pode ser conseguido pela aplicação de um papel molhado para o bloco, se necessário. Pode levar várias seções para atingir as condições de corte desejado para obter uma seção de qualidade. A seção é então flutuou em um banho de água e colocado sobre uma lâmina como em 16, supra. </li><li> Reagentes de coloração estão listados em Materiais abaixo. Protocolos são baseados em quatro Bancroft et al. Devido à variação de espessura é recomendado para otimizar manchas em lâminas. Rack de coloração dos tecidos pode provocar a flutuar fora do slide para adicionar a manchas diretamente para um slide plana pode ser necessária. Um protocolo idêntico ou coloração rack é necessário para resultados consistentes coloração exigida para histomorfometria. <ol class="lalpha"><li> Hematoxilina e eosina <ol class="lroman"><li> Mancha com Haematoxyilin 5-10 minutos </li><li> Lave bem com água da torneira Scott s </li><li> Mancha com Eosina 5 minutos </li><li> Lavar em água corrente </li><li> Limpar em xilol </li><li> Montar </li><li> Resultados <ol><li> Osteóide = rosa </li><li> Óssea calcificada = marrom arroxeada </li><li> Núcleos = blue </li></ol></li></ol></li><li> Von Kossa <ol class="lroman"><li> Lugar em solução de nitrato de prata e expor à luz forte até o osso fica preto (aprox. 10 min) </li><li> Lavar em água destilada por três vezes </li><li> Ameaça com tiossulfato de sódio por 5 minutos </li><li> Lavar em água destilada </li><li> Contracoloração com Safrinin O </li><li> Limpar em xilol </li><li> Montar </li><li> Resultados <ol><li> Óssea Mineralised = preto </li><li> Osteóide = vermelho / rosa </li></ol></li></ol></li><li> Masson Goldner s Trichrome <ol class="lroman"><li> Lavar com álcool alcalina (90mls de etanol 80% e 10mls de amônia 25% para 20 minutos) </li><li> Enxaguar em água </li><li> Enxágüe em água destilada </li><li> Mancha com Hematoxilina Weigert s por 10 minutos </li><li> Enxágüe em água destilada </li><li> Mancha com Ponceau-Fucsina solução final 5 minutos </li><li> Enxágüe com 1% de ácido acético 15 segundo </li><li> Mancha em fosfomolíbdico ácido-laranja solução G 5 minutos </li><li> Enxágüe com 1% de ácido acético 15 segundo </li><li> Mancha com luz verde 5 minutos </li><li> Enxágüe com 1% de ácido acético 3 mudanças </li><li> Enxágüe em água destilada </li><li> Montar </li><li> Resultados <ol><li> Óssea Mineralised = verde </li><li> Osteóide = vermelho / laranja </li><li> Núcleos = azul cinza </li><li> Cartilagem = roxo </li></ol></li></ol></li></ol></li><li> Preparações fluorocromo: </li><li> Estes são administrados por injecção intravenosa lenta, pelo menos duas semanas de intervalo. </li><li> Doses <ol class="lalpha"><li> Calceína Verde 10 mg / kg IV </li><li> Oxitetraciclina 50 mg / kg IV </li><li> Alizarina complexona 30 mg / kg IV </li></ol></li><li> Preparação de calceína Verde <ol class="lalpha"><li> Um grama de calceína Verde é titulado com NaOH 1 M até dissolver. </li><li> pH é ajustado com NaOH 1% até pH = 7,1-7,2. </li><li> Filtro e manter protegido da luz </li></ol></li><li> Preparação de Alizarina complexona <ol class="lalpha"><li> Três gramas de Alizarina complexona é titulado com NaOH 1 M até dissolver. </li><li> pH é ajustado com HCl 1% ou NaOH até pH = 7,1-7,2. </li><li> Filtro e manter protegido da luz </li></ol></li><li> Preparação de Oxitetraciclina <ol class="lalpha"><li> Oxitetraciclina está disponível como um fora da prateleira de drogas anti-microbial. Não requer preparação. </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>

Solventes: <ul><li> 10% normal Buffered ácido formalina, Xileno Etanol e Butanol Todos grau histológico, livre de enxofre. GRALE Scientific Pty Ltd. http://www.grale.com.au </li></ul> Moldes: <ul><li> Descascar, moldes de plástico GRALE Scientific </li></ul> Slides: <ul><li> Microscópio SuperFrost slides PLUS Lomb Scientific Pty Ltd. <a href="http://www.lomb.com.au/">http://www.lomb.com.au</a> </li></ul> Resina: <ul><li> Technovit GlycolMethacrylate 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> Fazendo bloco de resina-Technovit 3040 bloco Histo Heraeus Kulzer </li></ul> Manchas (Reagentes de GRALE Científica) Trichrome Goldner da mancha </li> 1. Ferro Weigert hemotoxylin (DeGroat) <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a) solução Hemotoxylin </td></tr><tr><td> Hemotoxylin </td><td> 1g </td></tr><tr><td> Álcool absoluto </td><td> 100mL </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) Solução de Ferro </td></tr><tr><td> 30% de cloreto férrico aquosa </td><td> 4mL </td></tr><tr><td> Ácido clorídrico (concentrado) </td><td> 1mL </td></tr><tr><td> água destilada </td><td> 95mL </td></tr></table> Filtrado e adicionado volume igual de hemotoxylin imediatamente antes mancha usado. 2. Ponceau-fucsina-azophloxin soluções estoque <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a) solução de Ponceau xilidina </td></tr><tr><td> Ponceau de xilidina </td><td> 0,75 g </td></tr><tr><td> fuchsion ácido </td><td> 0.25g </td></tr><tr><td> Ácido acético </td><td> 1mL </td></tr><tr><td colspan="2"> mix, e adicionar água destilada (100ml) </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) solução azophloxin </td></tr><tr><td> Azophloxin </td><td> 0.5g </td></tr><tr><td> Ácido acético </td><td> 0,6 ml </td></tr><tr><td colspan="2"> mix, e adicionar água destilada (100ml) </td></tr><tr><td colspan="2" style="font-weight:bold;"> c) Final solução de trabalho mancha </td></tr><tr><td> Ponceau-fucsina solução </td><td> 5-10ml </td></tr><tr><td> Azophloxin </td><td> 2mL </td></tr><tr><td> Solução de ácido acético 0,2% </td><td> 88mL </td></tr></table> 3. Solução Green Light <table style="margin-left:40px;" border="0"><tr><td> Luz verde </td><td> 1g </td></tr><tr><td> ácido acético </td><td> 1mL </td></tr><tr><td colspan="2"> Misture e adicione água destilada (500ml) </td></tr></table> 4. Phophomolybdic ácido-Orange solução G <table style="margin-left:40px;" border="0"><tr><td> Ácido fosfomolíbdico </td><td> 3g </td></tr><tr><td> Laranja G </td><td> 2g </td></tr><tr><td colspan="2"> Dissolver em 500mL de água destilada e adicionar timol cristal. </td></tr></table> Von Kossa Stain <table style="margin-left:40px;" border="0"><tr><td> 1% de nitrato de prata aquoso </td></tr><tr><td> Tiossulfato de sódio 2,5% </td></tr><tr><td> 1% de safranina O </td></tr></table> Alcian mancha azul <table style="margin-left:40px;" border="0"><tr><td> Alcian blue 8GX </td><td> 1g </td></tr><tr><td> Solução de ácido acético 3% </td><td> 100mL </td></tr></table> Hemotoxylin e eosina <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a) hemotoxylin Harris </td></tr><tr><td> Hemotoxylin </td><td> 2.5g </td></tr><tr><td> Álcool absoluto </td><td> 25mL </td></tr><tr><td> Alum de potássio </td><td> 50g </td></tr><tr><td> água destilada </td><td> 500mL </td></tr><tr><td> óxido de mercúrio </td><td> 1,25 g OR </td></tr><tr><td> Iodato de sódio </td><td> 0.5g </td></tr><tr><td> Ácido acético glacial </td><td> 20mL </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) solução de Eosina </td></tr><tr><td colspan="2"> Eosina produzido comercialmente foi usado. </td></tr></table> Todos os pós corante mancha são pós Gurr corante. Todos os reagentes são reagentes analíticos, e não Lab reagentes Fluorocromos <table border="1" cellspacing="0" cellpadding="0"><tr><td> Fabricante </td><td> Fluorocromo </td><td> Número Catálogo </td></tr><tr><td> Sigma-Aldrich, Sydney </td><td> Calceína Verde </td><td> C0875 </td></tr><tr><td> Sigma-Aldrich, Sydney </td><td> Alizarina complexona </td><td> A3882 </td></tr><tr><td> Intervet Austrália, Victoria </td><td> Oxitetraciclina (Fora da droga antimicrobiana prateleira) </td><td> Comercial; Engemycin 100 </td></tr></table>

undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis

Histologia óssea Undecalcified demonstra a micro-arquitetura do osso. Ele mostra tanto os componentes mineralizados e celulares do osso, que fornece informações vitais sobre o turnover ósseo ou formação e reabsorção óssea. Isto tem uma enorme importância em uma variedade de aplicações clínicas e de pesquisa. Ele produz belas imagens 1 e permite técnicas como a avaliação fluorocromo e histomorfometria 2. Análise fluorocromo é uma técnica onde corantes fluorescentes que se ligam ao cálcio são injetados em um ponto determinado momento, que permite a quantificação da quantidade de mineralização naquele momento. Histomorfometria é um processo de quantificação do osso ao nível microscópico. Realizar histologia óssea undecalcified é tecnicamente desafiador, especialmente com amostras de grande porte. Ela exige variações na técnica dos utilizados em parafina padrão incorporado histologia. Este vídeo ilustra o processo de produção de seções de boa qualidade e demonstra as dificuldades técnicas e métodos com os quais a superá-los. Preparação de amostras de fixação e processamento são alcançados com uma forma semelhante a outros tecidos moles, no entanto, devido à densidade e menor permeabilidade do osso consideravelmente maior fixação e os tempos de processamento são necessárias, muitas vezes levando várias semanas. Incorporação é alcançado usando um meio de suporte com dureza semelhantes ou iguais e densidade do osso, como metacrilato de resinas à base, mas ao contrário de infiltração de parafina e incorporação, este é um passo irreversível. Seccionamento pode ser conseguido através de moagem, que produz uma espessa seção, que é ótimo para os estudos, como a análise fluorocromo. Este é o melhor conseguido usando uma lâmina de diamante em uma macrotome. Alternativamente, as seções mais finas podem ser produzidas por microscopia de luz e isso é conseguido usando um micrótomo trenó com uma lâmina muito afiada. O micrótomo sledge fornece a maior resistência e estabilidade necessária para grandes blocos rígidos. Seções de resina incorporado pode ser manchado com uma variedade de manchas, que são demonstradas.

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

Histologia óssea Undecalcified fornece informações importantes para uma variedade de aplicações clínicas e de pesquisa. É tecnicamente desafiador, especialmente com amostras de grande porte. Este vídeo ilustra o processo de produção de seções de boa qualidade e demonstra as dificuldades técnicas e métodos com os quais a superá-los.

Preparação óssea Undecalcified de Histologia, Histomorfometria e Análise Fluorocromo

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

Universidad San Sebastian

Research

JoVE Journal

Biology

27.1K visualizações.  Monash University. Histologia óssea Undecalcified fornece informações importantes para uma variedade de aplicações clínicas e de pesquisa. É tecnicamente desafiador, especialmente com amostras de grande porte. Este vídeo ilustra o processo de produção de seções de boa qualidade e demonstra as dificuldades técnicas e métodos com os quais a superá-los.

Vídeo: 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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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

Biologia

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.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

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.

Preparação de adultos Drosophila Olhos de Seccionamento Fino e análise microscópica

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; Em situ</em&gt; Hibridação em embriões de Mount inteiros e histologia de células Adaptado para Elasmobrânquios Marinhos

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.

Osso meio condicionado: Preparação e Bioensaio

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.

Preparação de Amostras para Análise Mass Citometria

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.

Uma abordagem alternativa para preparação de amostras com baixo número de celular de Análise TEM

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.

Preparação da amostra para análise de Proteomics baseados em massa-espectrometria de Microvessels Ocular

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.

Um guia de processamento de imagens e secção, coring e imagem para aquisição e análise de amostras de ossos cortical de alto rendimento para micro-CT síncrotron

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.

Coleta de tecidos e extração de RNA da articulação do joelho osteoartrítica humana

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