Gli autori desiderano ringraziare Ikiel Ptito per il suo supporto tecnico. Siamo grati a Frank Ervin, Roberta Palmour e il personale del Behavioural Sciences Laboratories Fondazione situato a St Kitts, nelle Indie Occidentali, per il supporto continuo del nostro lavoro primati.

Parte 1: Pre-trattamento dei tessuti <ol><li> Tessuto deve essere ben perfusi con paraformaldeide, glutaraldeide, o formalina. Questo può essere raggiunto attraverso standard di perfusione transcardial tipicamente usato per raccogliere altri organi. Si raccomanda che poco dopo il sacrificio gli occhi da iniettare con fissativo appena sotto la lente e conservate in fissativo. </li><li> Nel presente studio il soggetto era profondamente sedato con cloridrato di ketamina (10 mg / kg, im), eutanasia con una dose eccessiva di sodio pentobarbital (25 mg / kg, iv) e perfusi transcardially con 0,1 M PBS fino a completo dissanguato. Questa è seguita da una soluzione di paraformaldeide al 4% in PBS per 5 min (~ 1 litro). </li></ol> Parte 2: rimozione del bulbo oculare dalla cavità orbitale <ol><li> Per facilitare l&#39;accesso al bulbo oculare, si raccomanda di rimuovere prima il cervello. Una volta che il cervello è stata rimossa la parete sottile osso orbita è evidente. Utilizzare il Rongeurs osso di chip lentamente la parete dell&#39;orbita. Tagliare i muscoli oculari con un bisturi e rimuovere il tessuto connettivo dal bulbo oculare. Tagliare con cautela il nervo ottico, questo può essere usato negli studi di microscopia elettronica. Il bulbo oculare dovrebbe essere rilasciato dalla cavità orbitale. </li></ol> Parte 3: Dissect la retina dalla oculare <ol><li> Posto l&#39;occhio in una piastra di Petri con PBS per mantenere la retina si secchi. Un microscopio da dissezione o tavolo montato ingrandimento stativo è utile nella dissezione, ma non una necessità. Togliere la cornea tagliando la sclera vicino al perimetro della cornea, a livello della serrata con un paio di forbici a molla e rimuovere l&#39;obiettivo con il forcipe. </li><li> Utilizzare un pennello, pinze, forbici e molla per rimuovere la retina dalla sclera. Questo viene fatto separando la retina dalla sclera e poi tagliando la sclera via con le forbici. Si deve svolgere questo processo con piccoli incrementi in modo da non danneggiare o strappare il tessuto retinico. La sclera non è facilmente separato dal resto del nervo ottico con tanta cura tagliare la sclera attorno al nervo ottico. </li><li> A questo punto della retina ha mantenuto la sua forma curva e deve essere appiattito per il campionamento. Prima di appiattimento della retina, il vitreo, che ha la consistenza di gelatina, possono essere rimosse in un grumo. Per appiattire la retina su un vetrino, fare parecchi tagli radiali con una lama di bisturi. Vitreo residuo può essere rimosso con ordinaria carta da filtro e un pennello. La strato di cellule gangliari della retina è esposta a questo punto quindi è imperativo essere gentile quando si rimuove il vitreo. Se il nervo ottico è ancora attaccato al disco ottico, togliere il nervo ottico, senza strappare la retina con un bisturi e un paio di forbici primavera. Questa è ora una retina montaggio flat (figura 1) e la fovea dovrebbe essere evidente come una macchia scura in direzione temporale / ventrale dal disco ottico. </li></ol> Parte 4: Campionamento <ol><li> Ci sono una serie di opzioni per il campionamento della retina. Qui descriveremo la preparazione flatmount e campionamento isodentric. Per entrambe le procedure flatmount la retina con lo strato in fibra ottica di distanza dalla presentazione. Se l&#39;intenzione è di esaminare la retina strato di cellule gangliari nella preparazione flatmount, è necessario rimuovere o candeggina l&#39;epitelio pigmentato. Per rimuovere l&#39;epitelio pigmentato usare un pennello leggermente per togliere una parte di questo livello, questa tende a danneggiare lo strato dei fotorecettori. Sbiancamento coinvolge ammollo la retina in soluzione di permanganato di potassio (0,25%) per 1 ora, il lavaggio in acqua distillata, e di compensazione in acido ossalico (5%) per 5 minuti 2. </li><li> Se l&#39;intenzione è di esaminare la distribuzione cellulare e la morfologia di ogni livello per tutta la retina, quindi si consiglia di campionamento isometrica della retina. La retina deve essere flatmounted su un vetrino e tenuto umido con PBS. Per il campionamento isometrica tagliare piccoli pezzi di tessuto equidistante dal disco ottico nel nasale, indicazioni temporali, inferiore e superiore (Figura 1). Non è necessario candeggina l&#39;epitelio pigmentato in questa preparazione. Questi pezzi possono ora essere sezionato nel piano coronale con un criostato, vibratome o ultramicrotomo a seconda della domanda di ricerca. Per sezionamento criostato, i pezzi devono essere crioprotetti nel 30% di saccarosio durante la notte e congelato nel contesto di un mezzo di montaggio in ghiaccio secco. In alternativa, i campioni possono essere inclusi in agar e fette su un vibratome. Il criostato e preparati vibratome sarà resa affidabile sezioni sottili come 4 m. Se sezioni più sottili sono necessari (ad esempio per la preparazione del microscopio elettronico), è necessario preparare i campioni per i ultramicrotomo. Per fare questo, le sezioni devono essere post-fissati in tetrossido di osmio per 1 ora sotto il fumehood. Ciò è seguita da disidratazione in una serie graduata di etanolo (50, 70, 95, 95, 100, 100%) e ossido di propilene 100%. Il tessutoviene poi incorporato in Epon (embed-812 kit di embedding). </li></ol> Parte 5: Risultati Rappresentante: Nel nostro laboratorio, abbiamo regolarmente eseguire immunoistochimica sulla retina cryosectioned (Figura 2). In questo caso siamo interessati alla isodensità dei recettori dei cannabinoidi (CB1) nella retina dei primati. Abbiamo anche esaminare gli effetti dell&#39;esposizione prenatale dell&#39;etanolo sul sistema visivo dei primati. A tal fine, siamo interessati a densità cellulare e lo spessore del livello nella fovea e nella retina periferica. Per fare questo, abbiamo incorporare pezzi di retina in Epon, fetta a 700 nm su un ultramicrotomo, e colorare con blu di toluidina 1% (Figura 3). <img src="/files/ftp_upload/1261/fig1.jpg" alt="figura 1" /> Figura 1 Retina Flatmount. Tagli radiali sono utilizzati per appiattire la retina. <img src="/files/ftp_upload/1261/fig2.jpg" alt="figura 2" /> Figura 2 immunocolorazione. Cryosection della retina periferica immunostained per CB1 dove le cellule gangliari della retina sono pesantemente etichettati con etichette negli strati interni ed esterni nucleare. Lo spessore di questa sezione è di 14 metri e colorato nella diapositiva. RG - strato retinico ganglio; IP - strato plessiforme interno; IN - interno strato nucleare; OP - strato esterno plessiformi; ON - strato esterno nucleare. <img src="/files/ftp_upload/1261/fig3.jpg" alt="figura 3" /> Figura 3 Fovea. Sezione attraverso la fovea che è stato incorporato in Epon e fette su un ultramicrotrome a 700nm. Lo strato dei fotorecettori (PR) è stato utilizzato per allineare le sezioni. Si noti che l&#39;intera estensione dei fotorecettori possono essere identificati indicando un angolo appropriato sul piano coronale. Misure di densità sono stati prelevati a 300 m, 500 m, e 800 m dal centro della fossa foveale utilizzando il sistema Bioquant Imaging. RG - strato retinico ganglio; IP - strato plessiforme interno; IN - interno strato nucleare; OP - strato esterno plessiformi; ON - strato esterno nucleare; barra di scala = 50 m

<ol>
	<li>Hendrickson, A., Kupfer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histogenesis+of+the+fovea+in+the+macaque+monkey.">The histogenesis of the fovea in the macaque monkey.</a> Invest Ophthalmol Vis Sci. 15, 746-756 (1976).</li><li>Herbin, M., Boire, D., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+distribution+of+retinal+ganglion+cells+in+the+St.+Kitts+green+monkey+(Cercopithecus+aethiops+sabeus).">Size and distribution of retinal ganglion cells in the St. Kitts green monkey (Cercopithecus aethiops sabeus).</a> J Comp Neurol. 383, 459-472 (1997).</li><li>Stone, 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> The Wholemount Handbook. , (1981).</li><li>Herbin, M., Boire, D., Theoret, H., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transneuronal+degeneration+of+retinal+ganglion+cells+in+early+hemispherectomized+monkeys.">Transneuronal degeneration of retinal ganglion cells in early hemispherectomized monkeys.</a> Neuroreport. 10, 1447-1452 (1999).</li><li>Krebs, W., Krebs, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primate Retina and Choroid Atlas of Fine Structure in Man and Monkey. , (1991).</li><li>Pow, D., Sullivan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+kinesis,+neurite+sprouting+and+abnormal+axonal+projections+of+cone+photoreceptors+in+the+aged+and+AMD-afflicted+human+retina.">Nuclear kinesis, neurite sprouting and abnormal axonal projections of cone photoreceptors in the aged and AMD-afflicted human retina.</a> Exp Eye Res. 84, 850-857 (2007).</li></ol>

La preparazione della retina come un wholemount permette l&#39;analisi della topografia e la distribuzione spaziale di uno strato di cellule gangliari o le cellule endoteliali dei vasi sanguigni della retina 3. Quantificazione della densità delle cellule nella periferia della retina dei primati è prontamente realizzato. Tuttavia, nelle regioni perifoveal e della fovea, la sovrapposizione degli strati multipli nello strato delle cellule gangliari ostacola quantificazione. Per aggirare questo pregiudizio pote...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Scalpel</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10003-12</td>
<td>&nbsp;</td>
<tr>
<td>Scalpel blades</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10011-00</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15020-15</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14090-11</td>
<td>Any surgical scissors are sufficient</td>
</tr>
<tr>
<td>Rongeurs</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>16121-14</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11027-12</td>
<td>&nbsp;</td>
<tr>
<td>Filter paper</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-924-150</td>
<td>&nbsp;</td>
<tr>
<td>Camel or Sable Hair paintbrush</td>
<td>&nbsp;</td>
<td>Art supply store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the gateway to the brain: dissecting the primate eye

Il sistema visivo nell&#39;uomo è considerata la porta verso il mondo e svolge un ruolo principale nella pletora di sensoriali, i processi percettivi e cognitivi. Non è quindi sorprendente che la qualità della visione è legata alla qualità della vita. Nonostante la diffusa ricerca clinica e di base che circonda le cause dei disturbi visivi, molte forme di disabilità visive, come la retinite pigmentosa e la degenerazione maculare, la mancanza di trattamenti efficaci. I primati non umani hanno la più vicina caratteristiche generali dello sviluppo dell&#39;occhio a quello umano. Non solo hanno un simile anatomia vascolare, ma tra gli altri mammiferi, i primati hanno la caratteristica unica di avere una regione nella retina temporale specializzata di alta acuità visiva, la fovea<sup&gt; 1</sup&gt;. Qui si descrive una tecnica generale per sezionare la retina dei primati per fornire il tessuto per esame istologico della retina, immunoistochimica, microdissezione laser capture, così come la luce e la microscopia elettronica. Con l&#39;ampio uso della primate non umano come modello traslazionale, la nostra speranza è che la migliore comprensione della retina forniranno approfondimenti approcci efficaci per attenuare o invertire gli effetti negativi di disturbi visivi sulla qualità della vita degli individui colpiti.

Watch this Scientific Journal Video about The Gateway to the Brain: Dissecting the Primate Eye at JoVE.com

The Gateway to the Brain: Dissecting the Primate Eye

Il primate non umano è una specie importante traslazionale per la nostra comprensione dello sviluppo e dell&#39;invecchiamento. L&#39;organizzazione anatomica della retina dei primati potrebbe fornire spunti importanti in condizioni normali e patologiche nell&#39;uomo.

La Porta del Cervello: Dissezione l&#39;Occhio Primate

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

the-gateway-to-the-brain-dissecting-the-primate-eye

Research

JoVE Journal

Biology

14.1K Views.  University of Montreal. Il primate non umano è una specie importante traslazionale per la nostra comprensione dello sviluppo e dell'invecchiamento. L'organizzazione anatomica della retina dei primati potrebbe fornire spunti importanti in condizioni normali e patologiche nell'uomo.

Video: The Gateway to the Brain: Dissecting the Primate Eye

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissezione e registrazione dal bivio C. Elegans neuromuscolare

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Ricerca

Biologia

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Lens trapianto in Zebrafish e la sua applicazione nell&#39;analisi dei Mutanti Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

Dissezione di non-umano cervello dei primati nello spazio Stereotassica

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

Conoscere ciò che conta: stereologia Tutti nel settore non umano cervello dei primati

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Un Reporter Novel RFP per aiutare nella visualizzazione del Disco Eye immaginale in Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Prelievo di sangue dalla vena della coda laterale del Topo

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Un tessuto metodo di compensazione per l'Imaging tridimensionale del tessuto adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Invertire la dissezione e dati DiceCT Reveal altrimenti nascosti nell'evoluzione del Primate viso

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

Un metodo ad alta uscita per isolare i periciti cerebrali dal mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Una guida passo-passo all'elettroantennography delle zanzare

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...