We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1。组织收获和肌肉组织的冷冻<ol><li>安乐死的小鼠用异氟烷（2-氯-2-（二氟甲氧基）-1,1,1-三氟乙烷）的过量。执行此步骤在通风橱中并利用一第二容器的方法，如干燥器或钟罩含有棉签浸泡在异氟醚。 </li><li>用力挤压脚垫确认死亡。使用安乐死的辅助方法，如颈椎脱位。 </li><li>湿用70％酒精的皮毛，以减少对肌肉松弛头发缕粘着。用细镊子，如＃5剥离皮肤脱落的肢体。 </li><li>小心地从骨骼中分离的兴趣（）在这种情况下，胫骨前（TA）的肌肉的下面，从肌腱开始并移除。 </li><li>切除的肌肉，它覆盖在华侨城（最佳切削温度化合物），并把它平放在一个标有一次性冻结模具。确保肌肉在其正常的生理方向（头至大l）在不拉伸。可替代地，冻结小发泡胶正方形肌肉和利用昆虫针以确保肌肉的正确长度针它。 </li><li>寒意2-甲基丁烷（异戊烷）中采用液氮钢制烧杯中。这平均需要3 - 5分钟。不断搅拌间歇直至白色沉淀开始形成在烧杯的底部和壁上。 注意:当这种情况发生时，异戊烷已达到最佳冷冻温度（-150℃） </li><li>浸含在冷冻2-甲基丁烷肌肉的模具。冷冻小肌肉，如隔膜和趾长伸肌（EDL）6 - 12秒，较大的肌肉如腓肠肌比目鱼（GS）和肱三头肌15 - 20秒。不要冷冻肌肉太久，因为这可能会导致开裂。 </li><li>转让的冷冻组织干冰，让异戊烷蒸发（注意，此过程平均需要大约15 - 20分钟）。包裹组织中铝箔并将其存储在üLTRA低温（-70℃〜-80℃），直到切片。 </li></ol> 2.嵌入组织在OCT准备组织块<ol><li>干冰运输组织，以cyrostat防止解冻。转移组织到低温恒温室（设定为-20至-24℃，以避免组织的充分解冻），并让他们平衡30分钟。 </li><li>打开珀耳帖冷却海湾上。如果低温恒温器不具备这种冷却，使用喷雾冷却喷雾快速冻结十月不要让肌肉解冻的任何一点的嵌入，因为这可能会导致冻害期间。 </li><li>添加均匀的薄层华侨城在试样上盘，让它冷却。当大多数的OCT被冻结在底部，同时仍然液体在顶部，插入在正确的方向的组织（垂直华侨城为横截面和平行华侨城为纵向部分）。使用喷雾冷却喷雾迅速冷冻十月触摸组织unembeded部分热转ractor并等待45秒。 </li><li>添加另一个薄层OCT的周围的肌肉，喷洒气溶胶冷却喷雾和提取的热量作为在上一步。 </li><li>在保持小幅度增加OCT和快速使用气溶胶喷雾冷却和加热萃取的结合冻结华侨城，直到整个肌肉被覆盖。 </li><li>放的热量提取的组织块的顶部，等待5分钟。 </li></ol> 3.准备部分和肌肉半山区肚识别章节<ol><li>安装试样到样品头。拧紧旋钮固定盘。确保在试样圆盘与所述样品头良好的接触。 </li><li>调节室内温度°之间-21和-24℃，等到设定温度达到。 </li><li>其厚度调节设置（7微米）;修剪通过推进或使用粗，细设置缩回块的块。 </li><li>收集部分通过按滑动的顶部上的部分中，使用相反电荷之间的吸引力，以方便切割部分以滑动的粘附预热带正电的显微镜载玻片。 </li><li>通过测定各部分的横截面面积（CSA）识别中期腹部分。连接到显微镜的计算机上执行此操作使用图像处理软件。 注:相衬图像的拍摄和整个肌肉的圆周经由上的软件程序跟踪功能测定。这将产生两个CSA以及最小鼬直径（测量纤维横截面尺寸的被表示为对一个粒子相反两侧的两个平行切线之间的最小距离）的测量。注意:当CSA和鼬最小直径测量开始高原，肚皮中旬已经达到。 </li><li>库存和归档的幻灯片在-80℃的未来染色。 注意:OCT不需要之前被除去以stainin摹程序​​。一旦载玻片盖上盖玻片，他们准备立即染色。 </li></ol> 4.染色注:虽然详细的一步一步的程序，可以在产品数据表中找到，并应遵循染色程序，个人优化可能需要以获得最佳的染色。 <ol><li>因为安装介质（见表）是不与水混溶的，通过分级醇增加脱水载玻片（每次2分钟，70％，95％和100％的乙醇），并清除它们用二甲苯（100％，5分钟），以确保幻灯片不会成为不透明随着时间的推移。 </li><li>盖玻片部分，并让他们干。图片显微镜配备图像处理软件。 </li></ol>

<ol>
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A., Kostek, C. A., Miller, J. 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The authors have nothing to disclose.

组织学和免疫组化已被用作主要的工具在理解表现和病理的进展在各种神经肌肉疾病。经典地，肌肉活检第一固定在甲醛，然后用石蜡包埋蜡。虽然甲醛固定保持组织结构，并允许组织存储和运输，在RT，这也灭活酶和蛋白质交联迫使进一步措施，以实现精确的免疫染色。这个问题与使用冰冻切片消除，但这种方法提出了获得最佳的染色效果和准确的诠释14,15独特的技术挑战。 <p class="j...

检查肌肉组织学中起着不同类型神经肌肉疾病1-4中的认识至关重要的作用。当与其他技术相结合，它使研究人员能够更好地了解病理的表现和进展的，也可以是必需评估治疗干预5-10的功效。然而，要准确，组织需要以适当的方式被保留以便切除之前立即保持生理状态。这种去除，保存，切片，和染色过程中需要非常小心。 组织可以以两种不同的方式进行光学显微镜研究来制备。第一种方法，福尔马林固定的石蜡包埋（FFPE）的部分，需要组织到被嵌入石蜡11,12之前被固定在10％的天然缓冲甲醛。固定步骤，有助于更好地保存形态，而且交联内源性PRoteins因而需要复杂的抗原修复程序用于免疫染色和消除酶染色对这些部分12的可能性。冰冻切片，另一方面，不需要进行预固定或处理;因此，蛋白质能保留原生生物构13,14。此外，冷冻切片也允许快速的周转时间，这有时是必要的，例如在肉瘤和其他外科活组织检查15的术中诊断。但是，如果没有正确完成，该方法是容易冻结损坏，它引入了改变肌肉结构使得部分不适宜用于组织学检查。本文将概述准备肌肉活检，以达到正确检查最佳染色最有效的方法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica CM 1850&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td>Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 
			Richard Allen Scientific</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse 50i&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Olympus</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Nikon Imaging Software Basic Research</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Image J, Metamorph</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>14043-220-05</td>
			<td>JD Medical Dist. Co., Inc.</td>
			<td>JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>4583</td>
			<td>Sakura Inc.</td>
			<td>Sakrua Fintek, USA, Torrence, CA 90501, USA 
			Leica Microsystem</td>
		</tr>
		<tr>
			<td>Freeze It</td>
			<td>23-022524</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Disposable tissue mold</td>
			<td>22-363-554</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Colorfrost plus slides</td>
			<td>9991001</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>650501-4L</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>HC-1300-1GL</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>M 32631-SL</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HC-7001GA</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Cytoseal 280</td>
			<td>8311-4</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Hematoxylin Gill #3</td>
			<td>CS402-1D</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>6766007</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
	</tbody>
</table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

肌肉活检组织学评价曾担任在神经肌肉疾病的病理发展过程的理解不可或缺的工具。然而，为了这样做，适当的照料需要切除并保存组织，以达到最佳的染色时，必须考虑。组织保存的一种方法包括固定在甲醛组织，然后用石蜡包埋它们。此方法保留形态良好，并且允许长期储存于RT但是麻烦的，并且需要处理的有毒化学品。此外，甲醛固定导致抗原的交联，这需要抗原修复协议有效免疫染色。与此相反，冷冻切片不需要固定，如此保留生物抗原的构象。此方法还提供了一个明显的优势在周围时间快转，使得它在情况需要像intraopera快速组织学评估特别有用略去手术活检。这里，我们描述制备肌肉活检用于可视化具有不同的组织学和免疫染色的最有效的方法。

该组向上收获和组织冷冻可以看出，在图1中 。切下的组织应存储在浸泡在PBS中，以避免在干燥（ 图1A）纱布。当组织被选择以用于组织学分析，他们应该被浸入OCT和布置在低温模具的正确方向，并尽可能接近生理长度尽可能以确保准确的形态（ 图1B）。异戊烷适于组织冷冻的浆液可以通过冷却的异戊烷在液氮中，并搅拌，直到小白色沉淀出现在底部来实现?...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

do-s-don-ts-preparation-muscle-cryosections-for-histological

Research

JoVE Journal

Medicine

31.7K 次观看.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

视频: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

科研

医学

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

在犬体内的肌肉功能测定

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

解剖的小鼠胰腺的组织学分析与代谢谱

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

超声心动图与小鼠心脏形态学的组织学检查

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...