Gli autori desiderano ringraziare per l&#39;aiuto della signora Sue Connell per la sua esperienza in embedding resina e la sig.ra Stefania Tombe per la sua esperienza di laboratorio. Gli autori desiderano ringraziare il professor Frank e il dottor Kandziora Marie-Anne Polboth per i loro consigli.

<ol><li> Il tessuto è inserito in un contenitore sigillato contenente il 10% tampone fosfato soluzione di formalina 3. Garantire la minima esposizione alla luce, se l&#39;analisi fluorocromo deve essere eseguita, in quanto il fluorocromi sono fotosensibili. </li><li> Stabilire che l&#39;orientamento del tessuto sarà richiesto. </li><li> Tagliare il campione a misura, con l&#39;orientamento corretto, utilizzando una sega a nastro. Adottare misure di sicurezza sono rispettate. Il campione viene poi inserito in un contenitore opaco, il cui volume dovrebbe essere circa dieci volte la dimensione del campione per ottenere una fissazione adeguata. Il campione dovrebbe rimanere nella soluzione di formalina per un totale compreso tra una e due settimane a seconda della sua grandezza. E &#39;suggerito di usare tessuto di controllo per i test e le finalità di ottimizzazione. </li><li> Controllare il tessuto si inserisce nello stampo embedding e posizionare il tessuto in un contenitore sigillato opaco contenente etanolo al 70%. </li><li> Processo il tessuto in modo crescente concentrazione di etanolo, schermatura dalla luce e sotto costante agitazione. <ol class="lalpha"><li> Etanolo al 70% 1 settimana </li><li> 80% di etanolo 1 settimana </li><li> 90% di etanolo 1 settimana </li><li> 100% di etanolo 1 settimana </li></ol></li><li> Il tessuto viene poi eliminato in butanolo per 1 settimana, ancora una volta schermatura dalla luce e sotto costante agitazione. </li><li> La densità della resina e le ossa devono essere attentamente abbinati. La densità della resina può essere alterata (vedi punto 8) e si consiglia di incorporare un esemplare primo test per garantire la giusta densità. Noi usiamo Technovit 7100 (Heraeus Kulzer) per microscopia ottica ed analisi fluorocromo. Se immunoistochimica è richiesta una resina alternativa deve essere usato come Technovit 9100 (Heraeus Kulzer). </li><li> La soluzione 7100 Technovit preparazione può essere miscelata con piccole aliquote della soluzione rammollimento fornito con il kit, se richiesto, ma a non più del 5% il volume di soluzione preparazione, nella nostra esperienza la soluzione di ammorbidimento è raramente necessaria. Parti uguali di etanolo assoluto e liquidi Technovit di base 7100 sono mescolati e applicati al tessuto come una pre-infiltrazione soluzione per 24 ore, riducendo al minimo l&#39;esposizione alla luce e con agitazione. </li><li> La soluzione di infiltrazione è poi costituita con 1 g di induritore I (= 1 borsa), che si scioglie in 100 ml di liquido base. Questo sostituisce il pre-infiltrazione soluzione ed è autorizzato a infiltrarsi per 1 settimana in agitazione costante. </li><li> Posizionare il tessuto nello stampo, con la faccia superficie di taglio verso il basso. Assicurarsi che vi sia un margine (idealmente almeno 5 millimetri) di tutto il tessuto per la resina, che assicura robustezza blocco. Mescolare 15ml di soluzione di preparazione Technovit 7100 con 1 ml di indurente II e versare attorno campione nello stampo e coprire di almeno 5 millimietres. Curare avrà inizio entro 2 ore, ma sarà completo durante la notte. </li><li> Montaggio viene quindi eseguita da modellare un blocco di supporto utilizzando Technovit 3040 (Heraeus Kulzer). Mix Technovit 3040 con un rapporto volume di 2 parti di polvere e 1 parte di liquido per ottenere un liquido viscoso. Versare Technovit 3040 nella rientranza sul retro del blocco ad un livello di almeno 2 mm al di sopra della base del blocco. Dopo circa 10 minuti lo stampo può essere staccato e il blocco è pronto. </li><li> La densità e quindi la proprietà di taglio della resina in metacrilato sono suscettibili di l&#39;umidità nella stanza. I blocchi devono essere conservati in un essiccatore. </li><li> Sezionamento terra produce grandi sezioni tra 20-50 micron di spessore. E &#39;utile per l&#39;analisi fluorocromo (vedere la sezione sotto), come più spesso sezioni produrre più luminoso fluorescenza. Fissare il disco diamantato per macrotome. Aggiungere lubrificante, come lo spirito di petrolio al serbatoio sul macrotome. Fissare il blocco nel morsetto e orientare a lama. Lasciare rettifica avvenga lentamente (circa 20-30 minuti per ogni sezione) garantire un&#39;adeguata lubrificazione. La prima sezione espone il volto di taglio e orienta la sezione, deve essere eliminato. </li><li> La sezione successiva è poi inserito in una diapositiva Superfrost Plus. La sezione ha la tendenza ad arricciarsi quindi si consiglia di mettere una parte di un sacchetto di panini su di esso e poi tenendola piatta con un altro scivolo bloccato in posizione. E &#39;poi inserito in un incubatore di tra 60-80 gradi centigradi per 1 ora. Questo ammorbidisce la metacrilato e aiuta la sezione aderire alla diapositiva in modo piatto. </li><li> La sezione a terra può essere pulito e lucidato, come richiesto con carta vetrata fine grana in modo gentile. </li><li> Il microtomo slitta tagli sezioni sottili, che fornisce le migliori sezioni per microsocopy luce. Utilizzare un microtomo slitta come ha aggiunto la rigidità, che è richiesto per il taglio di questi blocchi duri. In alternativa, un microtomo rotativo alimentato può essere utilizzato. Utilizzare una lama affilata in acciaio inossidabile. E &#39;preferibile avere a disposizione due lame come si può essere affilati come l&#39;altro viene utilizzato. La lama deve fare un angolo di 45 gradi con il blocco. Suammorbidimento rface può essere ottenuto applicando una carta bagnata al blocco, se necessario. Si può richiedere diverse sezioni per realizzare le condizioni di taglio desiderata per ottenere una sezione di qualità. La sezione è poi quotata in un bagno di acqua e posto su un vetrino come in 16 di cui sopra. </li><li> Reagenti di colorazione sono elencati in Materiali di seguito. Protocolli si basano su 4 Bancroft et al. A causa della variazione di spessore si consiglia di ottimizzare le macchie sui vetrini. Rack può causare colorazione dei tessuti a galleggiare fuori dalla diapositiva in modo da aggiungere le macchie direttamente a una diapositiva piatto può essere richiesta. Un protocollo identico o colorazione rack è necessario per esiti colorazione costante necessaria per istomorfometria. <ol class="lalpha"><li> Ematossilina ed eosina <ol class="lroman"><li> Macchia con Haematoxyilin 5-10 minuti </li><li> Lavare bene con acqua di rubinetto Scott s </li><li> Colorare con Eosina 5 minuti </li><li> Lavare in acqua di rubinetto </li><li> Chiara in xilene </li><li> Montare </li><li> Risultati <ol><li> Osteoide = rosa </li><li> Osso calcificato = bruno violaceo </li><li> Nuclei = blu </li></ol></li></ol></li><li> Von Kossa <ol class="lroman"><li> Posto in soluzione di nitrato d&#39;argento e di esporre alla luce forte fino a quando l&#39;osso mineralizzato diventa nero (circa 10 minuti) </li><li> Lavare in acqua distillata per tre volte </li><li> Minaccia con tiosolfato di sodio per 5 minuti </li><li> Lavare in acqua distillata </li><li> Contrasto con Safrinin O </li><li> Chiara in xilene </li><li> Montare </li><li> Risultati <ol><li> Osso mineralizzate = nero </li><li> Osteoide = rosso / rosa </li></ol></li></ol></li><li> Masson tricromica Goldner s <ol class="lroman"><li> Lavare con alcool alcalino (90mls del 80% etanolo e 10mls del 25% di ammoniaca per 20 minuti) </li><li> Risciacquare con acqua </li><li> Sciacquare con acqua distillata </li><li> Colorare con Ematossilina Weigert s per 10 minuti </li><li> Sciacquare con acqua distillata </li><li> Macchia con Ponceau-fucsina soluzione finale 5 minuti </li><li> Sciacquare con 1% di acido acetico 15 secondi </li><li> Colorare con fosfomolibdico acido-arancio soluzione G 5 minuti </li><li> Sciacquare con 1% di acido acetico 15 secondi </li><li> Macchia con la luce verde 5 minuti </li><li> Sciacquare con 1% di acido acetico 3 cambi </li><li> Sciacquare con acqua distillata </li><li> Montare </li><li> Risultati <ol><li> Osso mineralizzate = verde </li><li> Osteoide = rosso / arancio </li><li> Nuclei = blu grigio </li><li> Cartilagine = viola </li></ol></li></ol></li></ol></li><li> Preparazioni fluorocromo: </li><li> Questi sono somministrati mediante iniezione endovenosa lenta, di almeno due settimane di distanza. </li><li> Dosi <ol class="lalpha"><li> Calceina verde 10 mg / kg IV </li><li> Ossitetraciclina 50 mg / kg IV </li><li> Alizarina complessone 30 mg / kg IV </li></ol></li><li> Preparazione di calceina verde <ol class="lalpha"><li> Un grammo di calceina verde è titolato con NaOH 1 M fino alla dissoluzione. </li><li> pH è regolato con l&#39;1% NaOH fino a pH = 7,1-7,2. </li><li> Filtrare e tenere al riparo dalla luce </li></ol></li><li> Preparazione di alizarina complessone <ol class="lalpha"><li> Tre grammi di alizarina complessone è titolato con NaOH 1 M fino alla dissoluzione. </li><li> pH è quindi regolata con 1% di HCl o NaOH fino a pH = 7,1-7,2. </li><li> Filtrare e tenere al riparo dalla luce </li></ol></li><li> Preparazione del Ossitetraciclina <ol class="lalpha"><li> Ossitetraciclina è disponibile come al largo della piattaforma anti-microbico della droga. Non è necessaria la preparazione. </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>

Solventi: <ul><li> 10% Normale formalina tamponata, etanolo, xilene e butanolo acido Tutti grado istologico, senza zolfo. Grale scientifico Pty Ltd. http://www.grale.com.au </li></ul> Stampi: <ul><li> Staccarsi, stampi di plastica Grale scientifico </li></ul> Diapositive: <ul><li> SuperFrost PLUS Microscopio diapositive Lomb scientifico Pty Ltd. <a href="http://www.lomb.com.au/">http://www.lomb.com.au</a> </li></ul> Resina: <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> Backup blocco di resina Technovit 3040 blocco Histo Heraeus Kulzer </li></ul> Macchie (Reagenti da Grale scientifico) Goldner di colorazione tricromica </li> 1. Weigert ferro hemotoxylin (DeGroat) <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a) Hemotoxylin soluzione </td></tr><tr><td> Hemotoxylin </td><td> 1g </td></tr><tr><td> Alcol assoluto </td><td> 100 ml </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) Soluzione di ferro </td></tr><tr><td> 30% acquosa di cloruro ferrico </td><td> 4 ml </td></tr><tr><td> Acido cloridrico (concentrato) </td><td> 1 ml </td></tr><tr><td> Acqua distillata </td><td> 95mL </td></tr></table> Filtrato e aggiunto al volume uguale di hemotoxylin immediatamente prima macchia utilizzato. 2. Ponceau-fucsina-azophloxin magazzino soluzioni <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a) Ponceau de xilidina soluzione </td></tr><tr><td> Ponceau de xilidina </td><td> 0,75 g </td></tr><tr><td> acido fuchsion </td><td> 0,25 g </td></tr><tr><td> Acido acetico </td><td> 1 ml </td></tr><tr><td colspan="2"> mescolare e aggiungere acqua distillata (100ml) </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) azophloxin soluzione </td></tr><tr><td> Azophloxin </td><td> 0,5 g </td></tr><tr><td> L&#39;acido acetico </td><td> 0,6 ml </td></tr><tr><td colspan="2"> mescolare e aggiungere acqua distillata (100ml) </td></tr><tr><td colspan="2" style="font-weight:bold;"> c) Finale di lavoro soluzione macchia </td></tr><tr><td> Ponceau-fucsina soluzione </td><td> 5-10ml </td></tr><tr><td> Azophloxin </td><td> 2 ml </td></tr><tr><td> 0,2% soluzione di acido acetico </td><td> 88mL </td></tr></table> 3. Soluzione luce verde <table style="margin-left:40px;" border="0"><tr><td> Luce verde </td><td> 1g </td></tr><tr><td> acido acetico </td><td> 1 ml </td></tr><tr><td colspan="2"> Mescolare e aggiungere acqua distillata (500ml) </td></tr></table> 4. Phophomolybdic acido-Orange G soluzione <table style="margin-left:40px;" border="0"><tr><td> Fosfomolibdico acido </td><td> 3g </td></tr><tr><td> Arancio G </td><td> 2g </td></tr><tr><td colspan="2"> Sciogliere in 500 ml di acqua distillata e aggiungere timolo cristallo. </td></tr></table> Von Kossa Stain <table style="margin-left:40px;" border="0"><tr><td> 1% di nitrato d&#39;argento acquoso </td></tr><tr><td> Tiosolfato di sodio 2,5% </td></tr><tr><td> 1% safranina O </td></tr></table> Alcian macchia blu <table style="margin-left:40px;" border="0"><tr><td> Alcian blu 8GX </td><td> 1g </td></tr><tr><td> 3% di soluzione di acido acetico </td><td> 100 ml </td></tr></table> Hemotoxylin e eosina <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> Alcol assoluto </td><td> 25mL </td></tr><tr><td> Allume di potassio </td><td> 50g </td></tr><tr><td> acqua distillata </td><td> 500mL </td></tr><tr><td> ossido di mercurio </td><td> 1.25g O </td></tr><tr><td> Iodato di sodio </td><td> 0,5 g </td></tr><tr><td> Acido acetico glaciale </td><td> 20 mL </td></tr><tr><td colspan="2" style="font-weight:bold;"> b) Eosina soluzione </td></tr><tr><td colspan="2"> Eosina prodotto commercialmente è stato utilizzato. </td></tr></table> Tutte le polveri colorante macchia sono Gurr polveri coloranti. Tutti i reagenti sono reattivi e non reattivi di laboratorio Fluorocromi <table border="1" cellspacing="0" cellpadding="0"><tr><td> Fabbricante </td><td> Fluorocromo </td><td> Numero di catalogo </td></tr><tr><td> Sigma-Aldrich, Sydney </td><td> Calceina verde </td><td> C0875 </td></tr><tr><td> Sigma-Aldrich, Sydney </td><td> Alizarina complessone </td><td> A3882 </td></tr><tr><td> Intervet Australia, Victoria </td><td> Ossitetraciclina (Fuori della droga scaffale antimicrobici) </td><td> Nome commerciale; Engemycin 100 </td></tr></table>

undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis

Istologia ossea Undecalcified dimostra la micro-architettura del tessuto osseo. Esso mostra sia le componenti mineralizzata e cellulare di osso, che fornisce informazioni vitali sul turnover osseo o la formazione dell&#39;osso ed il riassorbimento. Ciò ha enorme importanza in una varietà di applicazioni cliniche e di ricerca. Si produce belle immagini 1 e consente di tecniche quali la valutazione fluorocromo e istomorfometria 2. Analisi fluorocromo è una tecnica in cui vengono iniettati coloranti fluorescenti che si legano al calcio in un punto particolare momento, che permette di quantificare la quantità di mineralizzazione in quel dato momento. Istomorfometria è un processo di quantificazione delle ossa a livello microscopico. Esecuzione di istologia undecalcified osso è tecnicamente impegnativo, soprattutto con esemplari di grandi dimensioni. Si richiede variazioni nella tecnica da quelli utilizzati in paraffina di serie incorporato istologia. Questo video illustra il processo di produzione di sezioni di buona qualità e dimostra le difficoltà tecniche e le modalità con le quali per il loro superamento. Preparazione dei campioni, fissazione e la lavorazione si ottengono con un modo simile a quello di altri tessuti molli, tuttavia a causa della densità e bassa permeabilità di osso molto più tempo di fissazione e tempi di lavorazione sono necessari, spesso prendendo diverse settimane. Incorporare si ottiene utilizzando un mezzo di supporto con durezza simili o uguali e la densità fino all&#39;osso come metacrilato, ma a differenza di infiltrazione di paraffina e l&#39;inclusione, questo è un passo irreversibile. Sezionamento può essere ottenuto dalla macinazione che produce una sezione più spessa, che è ottimale per gli studi, come l&#39;analisi fluorocromo. Questo è meglio realizzato utilizzando una lama di diamante su un macrotome. In alternativa, sezioni più sottili possono essere prodotti per la microscopia ottica e questo si ottiene utilizzando un microtomo slitta con una lama molto affilata. Il microtomo slitta fornisce la solidità e la stabilità necessaria per grandi blocchi duri. Resina sezioni incorporato può essere colorato con una varietà di macchie, di cui sia dimostrato.

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

Istologia ossea Undecalcified fornisce importanti informazioni per una varietà di applicazioni cliniche e di ricerca. E &#39;tecnicamente impegnativo, soprattutto con esemplari di grandi dimensioni. Questo video illustra il processo di produzione di sezioni di buona qualità e dimostra le difficoltà tecniche e le modalità con le quali per il loro superamento.

Bone Preparazione Undecalcified per Istologia, Istomorfometria e analisi 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 Views.  Monash University. Istologia ossea Undecalcified fornisce importanti informazioni per una varietà di applicazioni cliniche e di ricerca. E 'tecnicamente impegnativo, soprattutto con esemplari di grandi dimensioni. Questo video illustra il processo di produzione di sezioni di buona qualità e dimostra le difficoltà tecniche e le modalità con le quali per il loro superamento.

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.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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

Ricerca

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.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

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.

Preparazione di adulti Drosophila Occhi di sezionamento sottile e analisi microscopica

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; In situ</emIbridazione&gt; in embrioni Mount interi e istologia Adattato per Elasmobranchi Marine

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.

Bone mezzo condizionato: Preparazione e Bioassay

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.

Preparazione del campione per l&#39;analisi di massa 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.

Un approccio alternativo per la preparazione del campione con un basso numero di cellule per l&#39;analisi 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.

Preparazione del campione per l'analisi di proteomica massa-spettrometria-basato di microvasi oculari

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.

Guida all'elaborazione di sezioni, coring e immagini per l'approvvigionamento e l'analisi di campioni ossei corticali ad alta produttività per 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 ...

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.

Raccolta dei tessuti ed estrazione dell'RNA dall'articolazione del ginocchio osteoartritico umano

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