Die Autoren bedanken sich bei Ikiel Ptito für seine technische Unterstützung danken. Wir bedanken uns bei Frank Ervin, Roberta Palmour und das Personal des Behavioural Sciences Foundation Laboratories in St Kitts, West Indies, für ihre anhaltende Unterstützung unserer Primaten arbeiten.

Teil 1: Pre-Processing von Gewebe <ol><li> Tissue sollte gut mit Paraformaldehyd, Glutaraldehyd oder Formalin perfundiert werden. Dies kann durch Standard-transcardial Perfusion in der Regel verwendet, um andere Organe Ernte erreicht werden. Es wird empfohlen, dass kurz nach der Tötung der Augen mit Fixiermittel nur unter der Linse gespritzt werden und in Fixativ. </li><li> In der vorliegenden Studie das Thema war tief mit Ketaminhydrochlorid (10 mg / kg, im), mit einer Überdosis Natrium-Pentobarbital (25 mg / kg, iv) eingeschläfert sediert und perfundierten transkardial mit 0,1 M PBS, bis sie vollständig ausgeblutet. Dies wird durch eine 4% Paraformaldehyd-Lösung in PBS für 5 min (~ 1 Liter) folgten. </li></ol> Teil 2: Entfernung des Augapfels aus der Augenhöhle <ol><li> Für einen leichteren Zugang zu den Augapfel ist es empfehlenswert, entfernen Sie zunächst das Gehirn. Sobald das Gehirn entfernt wurde das dünnwandige Umlaufbahn Knochen ist leicht ersichtlich. Verwenden Sie die Knochen Rongeure langsam Chip entfernt die Wand der Augenhöhle. Schneiden Sie die Augenmuskeln mit einem Skalpell entfernen und das Bindegewebe aus dem Augapfel. Schneiden Sie vorsichtig den Sehnerv, kann diese in elektronischer Mikroskopie Studien verwendet werden. Der Augapfel wird jetzt aus der Augenhöhle freigegeben werden. </li></ol> Teil 3: Präparieren der Netzhaut von der Augenmuschel <ol><li> Legen Sie das Auge in eine Petrischale mit PBS auf die Netzhaut vor dem Austrocknen bewahren. Ein Binokular oder Tisch montiert Vergrößerungslampe Stand befindet sich in der Sektion nützlich, aber nicht eine Notwendigkeit. Entfernen der Hornhaut, indem die Lederhaut in engem Zusammenhang mit dem Umfang der Hornhaut auf der Ebene der Ora serrata mit einem Paar Frühling Schere und entfernen Sie das Objektiv mit der Zange. </li><li> Verwenden Sie einen Pinsel, Pinzette, Schere und Feder auf der Netzhaut von der Lederhaut zu entfernen. Dies ist durch die Trennung der Netzhaut von der Lederhaut und dann schneiden die Lederhaut weg mit der Schere gemacht. Man muss diesen Prozess auszuführen in kleinen Schritten, um nicht zu beschädigen oder zu reißen Netzhautgewebe. Die Sklera ist nicht ohne weiteres aus dem Rest des Sehnervs getrennt, so sorgfältig geschnitten der Lederhaut um den Sehnerv. </li><li> An diesem Punkt der Netzhaut hat ihre gebogene Form erhalten und muss für die Probenahme abgeflacht werden. Vor Abflachung der Netzhaut kann der Glaskörper, der die Konsistenz von Gelee hat, in einem Klumpen zu entfernen. Zum Reduzieren der Netzhaut auf eine Folie, um mehrere radiale Schnitte mit einem Skalpell. Die restlichen Glaskörper können nun mit gewöhnlichen Filterpapier und einem Pinsel entfernt werden. Die retinalen Ganglienzellen ist an dieser Stelle ausgesetzt so ist es unerlässlich, sanft zu sein, wenn das Entfernen des Glaskörpers ist. Wenn der Sehnerv ist noch in der Papille angebracht sind, entfernen den Sehnerv ohne zu reißen der Netzhaut mit einem Skalpell und ein Paar Frühling Schere. Dies ist nun eine flache Montage Netzhaut (Abbildung 1) und der Fovea sollte so ein dunkler Fleck in der zeitlichen / ventral von der Papille sichtbar. </li></ol> Teil 4: Probenahme <ol><li> Es gibt eine Reihe von Optionen zum Abtasten der Netzhaut. Hier beschreiben wir die Flatmount Vorbereitung und isodentric Probenahme. Für beide Verfahren Flatmount der Netzhaut mit den Glasfaser-Schicht von der Folie. Wenn die Absicht besteht, den retinalen Ganglienzellen in der Flatmount Vorbereitung zu untersuchen, ist es notwendig, entweder zu entfernen oder Bleichmittel das Pigmentepithel. So entfernen Sie das Pigmentepithel mit einem Pinsel leicht, einen Teil dieser Schicht zu entfernen, führt dies tendenziell zu den Photorezeptorschicht Schäden. Bleaching beinhaltet Einweichen der Netzhaut in Kaliumpermanganatlösung (0,25%) für 1 Stunde, Waschen in destilliertem Wasser, und das Clearing in Oxalsäure (5%) für 5 Minuten. 2 </li><li> Wenn die Absicht besteht, Zell-und Morphologie der einzelnen Schichten über die Netzhaut zu untersuchen, dann empfehlen wir isometrische Abtastung der Netzhaut. Die Netzhaut sollten auf einer Folie flatmounted und feucht gehalten mit PBS. Für isometrische Probenahme schneiden kleine Stücke von Gewebe gleich weit entfernt von der Papille in der Nase, zeitliche, oberen und unteren Richtungen (Abbildung 1). Es ist nicht notwendig zu bleichen das Pigmentepithel in dieser Zubereitung. Diese Stücke können jetzt in der koronalen Ebene geschnitten mit einem Kryostaten, Vibratom oder Ultramikrotom je nach Fragestellung. Für Kryostat Schnitte, sollten die Stücke in 30% Saccharose Nacht cryoprotected und eingefroren in Zusammenhang mit einem Eindeckmedium auf Trockeneis. Alternativ können die Proben in Agar eingebettet werden und in Scheiben geschnitten auf einem Vibratom. Der Kryostat und Vibratom Präparate zuverlässig Ertrag Abschnitte so dünn wie 4 m. Wenn dünner Schnitte erforderlich sind (z. B. für Elektronenmikroskop Vorbereitung), ist es notwendig, die Proben für die Ultramikrotom vorzubereiten. Um dies zu tun, sollten die Abschnitte werden post-fixed in Osmiumtetroxid für 1 Stunde unter dem Abzug durchzuführen. Dies wird durch Dehydrierung in einem abgestuften Ethanol-Reihe (50, 70, 95, 95, 100, 100%) und 100% Propylenoxid gefolgt. Das Gewebewird dann in Epon (Embed-812 Einbettung Kit) eingebettet. </li></ol> Teil 5: Repräsentative Ergebnisse: In unserem Labor haben wir routinemäßig Immunhistochemie auf kryogeschnitten retinae (Abb. 2). In diesem Fall sind wir in der isodensity von Cannabinoid-Rezeptoren (CB1) in der Netzhaut von Primaten interessieren. Wir untersuchen auch die Auswirkungen der pränatalen Ethanol-Exposition auf die Primaten visuellen Systems. Zu diesem Zweck sind wir daran interessiert, Zelldichte und Schichtdicke in der Fovea und in der peripheren Netzhaut. Um dies zu erreichen, betten wir Stücke von Netzhaut in Epon, Slice bei 700nm auf einem Ultramikrotom und Fleck mit 1% Toluidinblau (Abbildung 3). <img src="/files/ftp_upload/1261/fig1.jpg" alt="Abbildung 1" /> Abbildung 1 Flatmount Retina. Radial Schnitte werden verwendet, um die Netzhaut zu glätten. <img src="/files/ftp_upload/1261/fig2.jpg" alt="Abbildung 2" /> Abbildung 2 Immunfärbung. Gefrier der peripheren Netzhaut immungefärbt für CB1, wo die retinalen Ganglienzellen stark mit einigen Kennzeichnung in der inneren und äußeren nuklearen Schichten gekennzeichnet sind. Die Dicke dieser Abschnitt ist 14 m und gefärbt auf der Folie. RG - retinalen Ganglienzellen Schicht; IP - inneren plexiformen Schicht; IN - innere Körnerschicht; OP - plexiformen Schicht; ON - äußere Körnerschicht. <img src="/files/ftp_upload/1261/fig3.jpg" alt="Abbildung 3" /> Abbildung 3 Fovea. Schnitt durch die Fovea, die in Epon eingebettet war und in Scheiben geschnitten auf einem ultramicrotrome bei 700nm. Die Photorezeptorschicht (PR) wurde verwendet, um die Abschnitte auszurichten. Beachten Sie, dass das gesamte Ausmaß der Photorezeptoren identifiziert werden können zeigt einen geeigneten Winkel in der Frontalebene werden. Dichte-Messungen wurden bei 300 m, 500 m und 800 m vom Zentrum der Fovea Grube mit dem Bioquant Imaging-System übernommen. RG - retinalen Ganglienzellen Schicht; IP - inneren plexiformen Schicht; IN - innere Körnerschicht; OP - plexiformen Schicht; ON - äußere Körnerschicht; Balken = 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>

Die Vorbereitung der Netzhaut als wholemount ermöglicht die Analyse der Topographie und räumliche Verteilung der entweder die Ganglienzellschicht oder den Endothelzellen der retinalen Gefäße 3. Die Quantifizierung der Zelldichte in der Peripherie der Netzhaut von Primaten ist leicht erreicht. jedoch in perifovealen und fovealen Regionen, behindert das Stapeln mehrerer Schichten in den Ganglienzellen Quantifizierung. Um dies zu umgehen potenzielle Verzerrung kann der Fovea und perifovealen Region aus der w...

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the gateway to the brain: dissecting the primate eye

Das visuelle System des Menschen gilt als das Tor zur Welt und spielt eine Hauptrolle in der Fülle der sensorischen, perzeptiven und kognitiven Prozessen. Es ist daher nicht verwunderlich, dass die Qualität der Vision zur Verbesserung der Lebensqualität verbunden ist. Trotz der weit verbreiteten klinischen und der Grundlagenforschung über die Ursachen von Sehstörungen, fehlt es in vielen Formen von Sehbehinderungen, wie Retinitis pigmentosa und Makuladegeneration, wirksame Behandlungen. Nicht-menschliche Primaten haben die engste allgemeine Merkmale der Entwicklung des Auges zu der des Menschen. Nicht nur haben sie eine ähnliche vaskuläre Anatomie, aber unter anderen Säugetieren, haben Primaten die einzigartige Eigenschaft, die eine Region in der zeitlichen Netzhaut für eine hohe Sehschärfe, die Fovea spezialisiert<sup&gt; 1</sup&gt;. Hier beschreiben wir eine allgemeine Technik zum Präparieren der Primas Netzhaut um Gewebe für Netzhaut-Histologie, Immunhistochemie, Laser-Mikrodissektion, sowie Licht-und Elektronenmikroskopie liefern. Mit dem erweiterten Einsatz der nicht-menschlichen Primaten als translationale Modell ist unsere Hoffnung, dass ein besseres Verständnis der Netzhaut wird Einblicke in effektive Ansätze zur Abschwächung oder Umkehr der negativen Auswirkungen von Sehstörungen auf die Lebensqualität der betroffenen Personen liefern.

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

Die nicht-menschlichen Primaten ist ein wichtiger translationale Arten für unser Verständnis der Entwicklung und Alterung. Die anatomische Organisation des Primas Netzhaut kann wichtige Einblicke in normalen und pathologischen Bedingungen beim Menschen liefern.

Das Tor zum Gehirn: Die Biologie des Primas Eye

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 Aufrufe.  University of Montreal. Die nicht-menschlichen Primaten ist ein wichtiger translationale Arten für unser Verständnis der Entwicklung und Alterung. Die anatomische Organisation des Primas Netzhaut kann wichtige Einblicke in normalen und pathologischen Bedingungen beim Menschen liefern.

Serie: 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.

Dissecting und Aufnahme von C. elegans neuromuskulären Synapse

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

Forschung

Biologie

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.

Objektiv Transplantation in Zebrafisch und ihre Anwendung in der Analyse von Eye-Mutanten

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.

Grundlagen zur nicht-menschlichen Primaten Brain in stereotaktischen Raum

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.

Zu wissen, was zählt: Unbiased Stereology in der nicht-menschlichen Primaten Gehirn

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.

A Novel RFP Reporter in der Visualisierung des Eye Imaginalscheibe in Aid 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.

Blutprobenentnahme aus der laterale Schwanzvene der Ratte

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: Ein Gewebe Clearing-Methode für die dreidimensionale Darstellung von Fettgewebe

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.

Reverse-Dissektion und DiceCT offenbaren sonst versteckt Daten in der Evolution der Primaten-Gesicht

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.

Eine High Output-Methode zum Isolieren von zerebralen Pericyten von der Maus

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.

Eine Schritt-für-Schritt-Anleitung zur Mücken-Elektroantennographie

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 ...