저자는 자신의 기술 지원 Ikiel Ptito 감사하고 싶습니다. 우리는 프랭크 에르빈, 로버타 Palmour 및 영장류 작품들의 지속적인 지원 세인트 키츠에 위치한 Behavioural 과학 재단 연구소, 웨스트 인디스의 직원에게 감사하고 있습니다.

1 부 : 조직의 사전 처리 <ol><li> 조직은 잘 paraformaldehyde, 글루 타 알데히드, 또는 포르말린과 perfused해야합니다. 이것은 일반적으로 다른 장기를 수확하는 데 사용되는 표준 transcardial 재관류를 통해 달성될 수있다. 그것은 얼마 후이 눈이 그냥 렌즈에 따라 정착액과 주입과 정착액에 저장 희생하는 것이 좋습니다. </li><li> 현재 연구에서 주제가 깊이 나트륨 pentobarbital (25 MG / kg, IV)의 과다로 euthanized 케타민을 하이드로 클로라이드 (10 MG / kg, IM)과 진정했고 완전히 exsanguinated 때까지 0.1 M PBS로 transcardially perfused. 이것은 5 분 (~ 1리터)을 PBS에 4 % paraformaldehyde 솔루션옵니다. </li></ol> 2 부 : 궤도 구멍에서 안구 제거 <ol><li> 눈알을 쉽게하기 위해 그것은 먼저 두뇌를 제거하는 것이 좋습니다. 두뇌가 삭제되면 얇은 벽으로 둘러싸인 궤도 뼈가 쉽게 분명합니다. 서서히 궤도의 장벽을 멀리 칩에 뼈 rongeurs을 사용합니다. 메스로 안구 근육을 버려야하고 안구의 결합 조직을 제거합니다. 조심스럽게 시신경을 잘라, 이것은 전자 현미경 연구에서 사용할 수 있습니다. 눈알이 이제 궤도 캐비티에서 공개 되어야만합니다. </li></ol> 파트 3 : 세안 컵에서 망막을 해부하다 <ol><li> 건조에서 망막을 유지하기 위해 PBS와 페트리 접시에 눈을를 놓습니다. 해부 현미경 또는 테이블 조명 스탠드를 확대하는 것은 필요하지 해부에 유용하지만, 장착. 스프링 가위로 오라 세라타의 수준에있는 각막의 경계와 밀접하게 공막엔을 절삭하여 각막을 제거하고 포셉와 렌즈를 제거합니다. </li><li> 공막엔에서 망막을 제거하는 페인트, 포셉, 그리고 봄 가위를 사용합니다. 이것은 공막엔에서 망막을 분리 후 가위로 멀리 공막엔를 절단하여 이루어집니다. 하나는 작은 단위로있는만큼 손상이나 망막 조직을 찢어하지이 과정을 수행합니다. 공막엔는 쉽게 있도록 신중하게 시신경 주위 공막엔를 잘라 나머지 시신경에서 분리되지 않습니다. </li><li> 이 시점에서 망막의 곡선 모양을 유지하고 샘플링을 위해 평평해야합니다. 망막을 평평화 전에 젤리의 일관성을 가진 유리 유머는, 덩어리에서 제거할 수 있습니다. 슬라이드에 망막을 버리고, 메스 블레이드 여러 레이디얼 인하합니다. 잔여 유리체의 유머는 이제 일반 필터 종이와 페인트로 제거할 수 있습니다. 그것은 유리 유머를 제거하면 부드럽게하는 필수적인하므로 망막 신경절 세포 레이어는이 시점에서 노출됩니다. 시신경이 여전히 광학 디스크에 연결된 경우, 메스 칼날과 봄 가위를 사용하여 망막을 추출하지 않고 시신경을 제거합니다. 이것은 이제 평면 마운트 망막 (그림 1)이고 fovea는 광학 디스크에서 시간적 / 복부 방향으로 어두운 패치로 명확해야합니다. </li></ol> 4 부 : 샘플링 <ol><li> 망막을 샘플링 옵션이 많습니다. 여기서 우리는 flatmount 준비와 isodentric 샘플링을 설명합니다. 모두를위한 절차는 멀리 슬라이드에서 광섬유 레이어로 망막을 flatmount. 의도가 flatmount 준비에서 망막 신경절 세포 레이어를 조사하는 경우도 색소 상피를 제거하거나 표백하는 것이 필요합니다. 색소 상피이 레이어의 일부를 제거하는 가볍게 페인트를 사용하여 제거하려면,이 photoreceptor 레이어를 손상하는 경향이있다. 표백하면, 1 시간 동안 칼륨 과망간산염 솔루션 (0.25 %)에서 망막에 몸을 담글 수 증류수로 세척하고, 5 분 괭이밥에서 채취한 산성 (5 %)에서 지우면 포함됩니다. 2 </li><li> 의사가 망막을 통해 각 계층의 세포 분포 및 형태를 조사할 수있다면, 우리는 망막의 사시도 샘플링을 제안한다. 망막은 슬라이드에 flatmounted하고 PBS로 젖은 보관해야합니다. 사시 샘플링에 대한 콧소리, 현세의 상위 및 하위 경로 (그림 1)에있는 광학 디스크에서 등거리 조직의 작은 조각을 잘라 버릴거야. 이런 준비에 표백제 색소 상피를 필요는 없습니다. 이 작품은 현재 연구 질문에 따라 그라 이오 스탯, vibratome 또는 ultramicrotome와 코로나 비행기에서 sectioned 수 있습니다. 그라 이오 스탯의 sectioning 들어, 조각은 야간 30 % 자당에 cryoprotected이어야하며 드라이 아이스에 장착 매체의 맥락에서 냉동. 또한, 샘플은 한천에 포함된하고 vibratome에 얇게하실 수 있습니다. 그라 이오 스탯 및 vibratome 준비가 안정적으로 4 미터만큼 얇은 부분을 얻을 것입니다 얇은 섹션 (전자 현미경의 준비 등) 필요한 경우, 그것은 ultramicrotome에 대한 샘플을 준비하는 것이 필요합니다. 이렇게하려면 섹션 fumehood에 따라 1 시간 오스뮴 tetroxide에 포스트 수정되어야합니다. 이 등급 에탄올 탈수 시리즈 (50, 70, 95, 95, 100, 100 %) 및 ​​100 %의 프로필렌 산화물옵니다. 조직다음 Epon (임베드 - 812 포함 키트)에 포함된 것입니다. </li></ol> 제 5 부 : 대표 결과 : 저희 연구실에서는, 우리는 정기적으로 cryosectioned retinae (그림 2)에 immunohistochemistry을 수행합니다. 이 경우에는 우리는 영장류 망막에서 cannabinoid 수용체 (CB1)의 isodensity에 관심이 있습니다. 우리는 또한 영장류 시각 시스템에 태아 에탄올 노출의 효과를 검사합니다. 이를 위해, 우리는 fovea과 주변 망막에있는 세포 밀도와 레이어의 두께에 관심이 있습니다. 이것을 달성하기 위해, 우리는 ultramicrotome에 700nm에서, Epon에 슬라이스를 망막의 조각을 포함,, 1 % toluidine 블루 (그림 3)와 얼룩. <img src="/files/ftp_upload/1261/fig1.jpg" alt="그림 1" /> 그림 1 Flatmount의 망막. 레이디얼 상처는 망막을 평평하게하는 데 사용됩니다. <img src="/files/ftp_upload/1261/fig2.jpg" alt="그림 2" /> 그림 2 Immunostaining가. 주변 망막의 Cryosection는 망막 신경절 세포가 많이 안쪽과 바깥 핵 층의 일부 레이블과 레이블 아르 CB1에 대한 immunostained. 이 섹션의 두께는 슬라이드에서 14m와 얼룩이다. RG - 망막 신경절 층, IP - 내부 plexiform 층, IN - 내부 핵 계층, OP - 외부 plexiform 층, ON - 바깥 핵 층. <img src="/files/ftp_upload/1261/fig3.jpg" alt="그림 3" /> Epon에 포함된하여 700nm에서 ultramicrotrome에 얇게 있던 fovea을 통해 그림 3 Fovea. 절을 참조하십시오. photoreceptor 층 (PR)이 섹션을 정렬하는 데 사용되었다. photoreceptors의 전체 범위가 코로나 비행기에 적절한 각도를 나타내는 확인할 수 있습니다납니다. 밀도 측정은 300m, 500m, 그리고 Bioquant 이미징 시스템을 사용 foveal 구덩이의 중심에서 800m에서 찍은. RG - 망막 신경절 층, IP - 내부 plexiform 층, IN - 내부 핵 계층, OP - 외부 plexiform 층, ON - 바깥 핵 층, 규모 표시줄 = 50m

<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>

wholemount로 망막의 준비는 지형과 신경절 세포 레이어 또는 망막 혈관 3 내피 세포 중의 공간적 분포의 분석을 위해 수 있습니다. 영장류 망막의 주변에있는 세포 밀도 부량이 쉽게 수행됩니다. 그러나 perifoveal 및 foveal 지역에서 신경절 세포 레이어에 여러 레이어 스태킹은 부량을 방해. 이러한 잠재적인 편견을 회피하려면, fovea 및 perifoveal 지역은 코로나 비행기 2,4 년 반 얇은 섹션...

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the gateway to the brain: dissecting the primate eye

인간의 시각 시스템은 세계의 관문으로 간주하고 감각, 지각 및인지 과정의 과다에 주요 역할을합니다. 그것은 비전의 품질은 삶의 질을 묶어 것을 따라서 놀라운 일이 아닙니다. 시각 장애의 원인을 둘러싼 광범위한 임상 및 기초 연구에도 불구하고, 그러한 retinitis pigmentosa과 황반 변성 등 시각 장애, 여러 형태의 효과적인 치료를 부족합니다. 비 인간 영장류는 인간의 것과 눈 개발의 가장 일반적인 기능을 가지고 있습니다. 뿐만 아니라 그들은 유사한 혈관 해부학을 가지고 있지만, 다른 포유 동물 사이, 영장류가 높은 영상 시력, fovea에 대한 전문 시간적 망막에서 영역을 갖는 독특한 특성을 가지고 할<sup&gt; 1</sup&gt;. 여기 우리는 망막 조직학, immunohistochemistry, 레이저 캡처 microdissection뿐만 아니라, 빛과 전자 현미경을위한 조직을 제공하는 영장류 망막을 해부에 대한 일반적인 기술을 설명합니다. translational 모델로 비 인간 영장류의 확장 사용과 함께, 우리의 희망은 망막의 개선 이해가 영향을받는 개인의 삶의 질을에 시각 장애의 부정적인 영향을 attenuating 또는 반대 방향으로 효과적인 접근 방법에 대한 통찰력을 제공할 것입니다.

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

비 인간 영장류가 개발 및 노화에 대한 우리의 이해를위한 중요한 translational 수종이다. 영장류 망막의 해부 학적 조직은 인간의 정상 및 병리 학적 조건에 중요한 통찰력을 제공할 수 있습니다.

두뇌에 게이트웨이 : 프리 메이트 &#39;눈&#39;을 분석 해 봅시다

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 ...

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14.1K 조회수.  University of Montreal. 비 인간 영장류가 개발 및 노화에 대한 우리의 이해를위한 중요한 translational 수종이다. 영장류 망막의 해부 학적 조직은 인간의 정상 및 병리 학적 조건에 중요한 통찰력을 제공할 수 있습니다.

비디오: 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.

해부 및 C. 엘레간스 신경근육학 융티온에서 레코딩

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

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생물학

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.

렌즈 Zebrafish의 이식과 눈 돌연변이의 분석에의 응용

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.

Stereotaxic 공간에 비 인간 메이트 브레인 해부

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.

비 - 인간 메이트 두뇌의 편견 Stereology를 : 중요 한거지 아는

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.

에있는 아이 Imaginal 디스크의 시각화에 도움을 소설 RFP 리포터 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.

쥐의 측면 꼬리 정맥에서 혈액을 샘플링

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-지우기: 조직 지방 조직의 3 차원 이미징 방법 삭제

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.

해 부와 영장류의 진화에 DiceCT 공개 그렇지 않으면 숨겨진 데이터

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.

마우스에서 대뇌 골후혈구를 분리하는 고출력 방법

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.

모기 전기 조영술에 대한 단계별 가이드

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 ...