Os autores gostariam de agradecer Ikiel Ptito por seu apoio técnico. Somos gratos a Frank Ervin, Palmour Roberta e ao pessoal da Fundação Ciências Comportamentais Laboratories localizado em São Cristóvão, West Indies, por seu apoio contínuo do nosso trabalho de primatas.

Parte 1: Pré-processamento de tecido <ol><li> Tecido deve ser bem perfundidos com paraformaldeído, glutaraldeído ou formalina. Isto pode ser conseguido através de perfusão transcardial padrão normalmente utilizado para a colheita de outros órgãos. Recomenda-se que logo após o sacrifício dos olhos ser injetado com o fixador apenas sob a lente e armazenados em fixador. </li><li> No presente estudo o assunto estava profundamente sedado com cloridrato de cetamina (10 mg / kg, im), sacrificados com uma overdose de pentobarbital sódico (25 mg / kg, iv) e perfundidos transcardially com 0,1 M PBS até que esteja completamente sangrar. Isso é seguido por uma solução de paraformaldeído 4% em PBS por 5 min (~ 1 litro). </li></ol> Parte 2: A remoção do globo ocular da cavidade orbital <ol><li> Para facilitar o acesso ao globo ocular recomenda-se remover primeiro o cérebro. Uma vez que o cérebro foi removido o osso da órbita de paredes finas é facilmente perceptível. Usar o fórceps osso para roer lentamente a parede da órbita. Cortaram os músculos oculares com um bisturi e remover o tecido conectivo do globo ocular. Corte cuidadosamente o nervo óptico, este pode ser utilizado em estudos de microscopia eletrônica. O globo ocular deve agora ser lançado a partir da cavidade orbital. </li></ol> Parte 3: Dissecar a retina ocular do <ol><li> Colocar o olho em uma placa de Petri com PBS para manter a retina de secagem. Um microscópio de dissecação ou mesa montada ampliação suportar a luz é útil para a dissecação, mas não uma necessidade. Remova a córnea, cortando a esclera de perto para o perímetro da córnea ao nível da ora serrata com um par de tesouras primavera e remover a lente com a pinça. </li><li> Use um pincel, pinça, tesoura e mola para remover a retina da esclera. Isto é feito através da separação da retina da esclera e depois corte a esclera acabar com a tesoura. Deve-se realizar este processo em pequenos incrementos de modo a não danificar ou rasgar o tecido da retina. A esclera não é facilmente separado do nervo óptico remanescente tão cuidadosamente cortar a esclera em torno do nervo óptico. </li><li> Neste ponto, a retina manteve sua forma curva e precisa ser aplainado para a amostragem. Antes de achatamento da retina, o humor vítreo, que tem a consistência de geléia, podem ser removidos em um caroço. Para achatar a retina em um slide, fazer vários cortes radiais com uma lâmina de bisturi. O humor vítreo residual pode agora ser removido com papel de filtro comum e um pincel. A camada de células ganglionares da retina é exposta neste momento por isso é imperativo para ser gentil ao remover o humor vítreo. Se o nervo óptico ainda está ligado ao disco óptico, remova o nervo óptico sem rasgar a retina usando uma lâmina de bisturi e uma tesoura de mola. Esta é agora uma retina montagem plana (Figura 1) e fóvea deve ser aparente como uma mancha escura na direção temporal / ventral do disco óptico. </li></ol> Parte 4: Amostragem <ol><li> Há uma série de opções para a amostragem da retina. Aqui vamos descrever a preparação e amostragem flatmount isodentric. Para ambos os procedimentos flatmount da retina com a camada de fibra óptica de distância do slide. Se a intenção é examinar a camada de células ganglionares da retina na preparação flatmount, é necessário remover ou lixívia do epitélio pigmentado. Para remover o epitélio pigmentado use um pincel levemente para remover alguma desta camada, esta tende a danificar a camada de fotorreceptores. Clareamento envolve embeber a retina em solução de permanganato de potássio (0,25%) por 1 hora, lavar em água destilada, e limpando em ácido oxálico (5%) por 5 minutos. 2 </li><li> Se a intenção é examinar a distribuição de células e morfologia de cada camada em todo o retina, então sugerimos amostragem isométrica da retina. A retina deve ser flatmounted em um slide e mantido úmido com PBS. Para a amostragem isométrica cortar pequenos pedaços de tecido eqüidistantes do disco óptico no nasal, direções temporal, superior e inferior (Figura 1). Não é necessário para branquear a epitélio pigmentado nesta preparação. Essas peças podem agora ser seccionado no plano coronal com um criostato, vibratome, ou ultramicrótomo dependendo da questão de pesquisa. Para corte em criostato, as peças devem ser cryoprotected em sacarose 30% durante a noite e congelados no contexto de um meio de montagem em gelo seco. Alternativamente, as amostras podem ser incorporados em agar e cortados em um vibratome. O criostato e preparações vibratome confiantemente rendimento seções finas como 4 m. Se são necessários cortes mais finos (por exemplo, para a preparação de microscópio eletrônico), é necessário preparar as amostras para o ultramicrótomo. Para fazer isso, as seções devem ser pós-fixados em tetróxido de ósmio por 1 hora sob a fumehood. Isto é seguido por desidratação em uma série de etanol classificados (50, 70, 95, 95, 100, 100%) e óxido de propileno 100%. O tecidoé então incorporado em Epon (incorporar-812 kit incorporação). </li></ol> Parte 5: Resultados Representante: Em nosso laboratório, nós rotineiramente imunohistoquímica na retina cryosectioned (Figura 2). Neste caso, estamos interessados ​​na isodensity dos receptores de canabinóides (CB1) na retina de primatas. Também examinamos os efeitos da exposição pré-natal de etanol no sistema de primatas visual. Para este fim, estamos interessados ​​na densidade de células ea espessura da camada na fóvea e na retina periférica. Para conseguir isso, nós incorporar pedaços de retina em Epon, fatia a 700nm em um ultramicrótomo, e manchar com 1% de azul de toluidina (Figura 3). <img src="/files/ftp_upload/1261/fig1.jpg" alt="figura 1" /> Figura Retina Flatmount 1. Cortes Radial são usados ​​para achatar a retina. <img src="/files/ftp_upload/1261/fig2.jpg" alt="figura 2" /> Figura imunocoloração 2. Cryosection da retina periférica histoquímica para CB1 onde as células ganglionares da retina são fortemente marcadas com alguma rotulagem nas camadas interiores e exteriores nuclear. A espessura desta seção é de 14 m e coradas no slide. RG - camada ganglionares da retina; IP - camada plexiforme interna; IN - camada nuclear interna; OP - camada plexiforme externa; ON - camada nuclear externa. <img src="/files/ftp_upload/1261/fig3.jpg" alt="figura 3" /> Figura 3 Fovea Seção. Através da fóvea que foi incorporado em Epon e cortados em um ultramicrotrome em 700nm. A camada de fotorreceptores (PR) foi usado para alinhar as seções. Observe que a extensão total dos fotorreceptores pode ser identificado, indicando um ângulo apropriado no plano coronal. Medidas de densidade foram realizadas em 300 m, 500 m e 800 m do centro do poço foveal usando o sistema de imagem BIOQUANT. RG - camada ganglionares da retina; IP - camada plexiforme interna; IN - camada nuclear interna; OP - camada plexiforme externa; ON - camada nuclear externa; barra de escala = 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>

A preparação da retina como um wholemount permite a análise da topografia e distribuição espacial de uma camada de células do gânglio ou as células endoteliais dos vasos sanguíneos da retina 3. Quantificação da densidade de células na periferia da retina de primatas é facilmente realizado. No entanto, em regiões perifoveal e foveal, o empilhamento de múltiplas camadas na camada de células ganglionares obstrui quantificação. Para contornar esse viés potencial, a fóvea e região perifoveal p...

<!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

O sistema visual em humanos é considerada a porta de entrada para o mundo e desempenha um papel principal na infinidade de sentidos, processos perceptivos e cognitivos. Portanto, não é surpreendente que a qualidade de visão está ligada à qualidade de vida. Apesar da pesquisa clínica e básica generalizada sobre as causas de distúrbios visuais, muitas formas de deficiência visual, tais como retinite pigmentosa e degeneração macular, a falta de tratamentos eficazes. Primatas não-humanos têm o mais próximo características gerais do desenvolvimento do olho ao dos humanos. Não só eles têm uma anatomia semelhante vascular, mas entre outros mamíferos, os primatas têm a característica única de ter uma região na retina temporal especializados para a acuidade visual de alta, a fóvea<sup&gt; 1</sup&gt;. Aqui nós descrevemos uma técnica geral para dissecção da retina de primatas para fornecer tecido para histologia da retina, imuno-histoquímica, microdissecção de captura de laser, assim como a luz e microscopia eletrônica. Com o uso prolongado do primata não-humano como um modelo de translação, a nossa esperança é que uma melhor compreensão da retina irá fornecer insights sobre abordagens eficazes para atenuar ou reverter o impacto negativo dos distúrbios visuais na qualidade de vida dos indivíduos afetados.

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

O primata não-humano é uma importante espécie de translação para a nossa compreensão do desenvolvimento e envelhecimento. A organização anatômica da retina de primatas pode fornecer importantes insights sobre condições normais e patológicas nos seres humanos.

A entrada para o cérebro: Dissecando o olho Primaz

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 visualizações.  University of Montreal. O primata não-humano é uma importante espécie de translação para a nossa compreensão do desenvolvimento e envelhecimento. A organização anatômica da retina de primatas pode fornecer importantes insights sobre condições normais e patológicas nos seres humanos.

Vídeo: 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.

Dissecação e gravação a partir da junção neuromuscular C. Elegans

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

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.

Transplante de lente em Zebrafish e sua Aplicação na Análise de Mutantes 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.

Dissecando o cérebro do primata não-humano no espaço estereotáxico

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.

Sabendo o que conta: Estereologia Imparcial no cérebro de primatas não humanos

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 Reporter RFP para ajudar na visualização do Disco Imaginal Eye em 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.

A amostragem de sangue a partir da veia lateral da cauda do rato

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: Um método de compensação por imagem tridimensional do tecido adiposo tecido

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.

Reverter a dissecção e dados DiceCT revelar outra forma escondida na evolução do primata rosto

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.

Um método de alta saída para isolar pericites cerebrais do rato

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.

Um guia passo-a-passo para eletroantennografia de mosquitos

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 ...