These experiments were funded by the NIH grant #RAI093634A. We express immense gratitude to Jenny Powers, DVM, PhD, for her assistance in muntjac milking.

1，鼠标挤奶协议在本协议中所述的方法进行批准IACUC。 1，分离大坝的后代<ol><li>选择要挤奶的大坝。选择具有4个或更多的小狗一胎是8-12天分娩后提供最大收奶，虽然集合可以在任何时间点分娩后长达21天的大坝。 </li><li>挤奶前，从她的垃圾至少2小时分开大坝。 注：如果出现一种愿望，牛奶多个水坝在给定的时间内，是可以接受的房子在同一个笼子里分离出的大坝有足够的食物和水的供应。 </li></ol>催产素，麻醉和眼部润滑剂2。管理<ol><li>辖2个国际单位/公斤腹腔注射催产素（IP）。催产素是一种激素，作用于女性哺乳期的乳腺，刺激乳汁的释放。催产素可以被收购FROM一名兽医。 </li><li>管理含有80-100毫克/千克氯胺酮在5-10毫克/公斤甲苯噻嗪IP相结合的麻醉剂。例如，一个10毫升储麻醉剂可以提前做好准备对小鼠体重约30g，并将包括0.1毫升甲苯噻嗪，8.9毫升水2 O和1.0毫升氯胺酮。 0.25毫升总麻药会沉着鼠标这种规模的约20〜30分钟，应提供足够的时间，收奶。 注：要确定该动物已成功麻醉后，轻轻地放在她在她的肚子上的固体表面;她不应该动她的脚或试图上升到她的脚，如果她是完全麻醉。在鼠标未充分与原剂量麻醉的情况下，研究者可以管理另一¼或麻醉剂混合物取决于鼠标和/或镇静的水平，该小鼠已经实现的尺寸的原始剂量的半。然而，这我能ncrease不良反应麻醉的机会，并且还可能会导致鼠标被更深度麻醉和/或麻醉的时间较长。 注意：如果挤奶多​​于一个坝，就必须错开催产素和由于催产素和镇静时间氯胺酮和甲苯噻嗪的半衰期短，麻醉剂注射。 </li><li>取少量眼部润滑剂，以每只眼睛的角落和蔓延，防止老鼠的眼睛干涩，同时在麻醉下。 </li></ol> 3，牛奶收集奶源是最容易进行的两个研究人员：一名研究人员，以保持麻醉的小鼠，而手动表达的牛奶（简称R1），和一个研究人员收集的牛奶（R2）。这种方法也可以与一个人进行的，如果鼠标被固定在平坦表面上，以避免伤害。 <ol><li> R2应配备消毒酒精消毒片，一个P-200点子etman，干净的移液管尖用于每个小鼠，和一个容器来保存牛奶，如在1.5 ml Eppendorf管或一个1.2毫升离心管（见表材料）。 </li><li>由于催产素开始生效，放乳将成为老鼠的乳腺区可见（ 见图1A）。 R1：拆开消毒酒精棉片轻轻擦拭鼠标的乳腺区域之前，挤奶清洁的区域。使用拇指和食指轻轻按摩和挤压乳腺组织在向上运动，直到牛奶可见珠开始形成于乳头基部手动挤出乳汁从乳头。 </li><li>按P-200 PIPETMAN柱塞其第一站，释放出空气的枪头和牛奶收集准备。 </li><li>定位移液管尖端处的乳滴的顶部，并通过缓慢释放活塞上的压力轻轻拉动牛奶进入移液管尖端。注意不要把尖太靠近乳头或皮肤（见图1B）。它没有必要完全释放所述柱塞，因为这将拉动牛奶一路枪头的顶部，并会使得难以排出常粘稠奶到保持容器。 </li><li>通过使用拇指按压柱塞过去的第一站（ 见图1C）排出乳汁进入容器。为了便于排出小体积，压在管的一侧的移液管尖端，使得没有液体就会丢失。 </li><li>继续从每个乳头人工挤奶，朝着顺时针或反时针方向，以便不跳过任何奶头。它既是可以接受的，有用回来，并不断表达的牛奶来自同一个乳头是否应该产生比另一个更牛奶。 </li></ol>在大坝4。麻醉逆转<ol><li>在大多数情况下，它是很常见的大坝开始，开始在挤奶过程中醒来。如果不是这种情况，将大坝的CAGË间接热源，如加热循环毯子，下方直到她开始醒来。 注：由于大坝开始醒来，密切观察她的活动，直到她能走约她在她自己的笼子里。 </li><li>一旦大坝走动笼而不争，把她放回她原来的笼子里与她的垃圾。没有不良影响已经注意到对于幼崽“收奶后，喂能力。 </li></ol> 2，麂挤奶协议在本协议中所述的方法进行批准IACUC。 1，分离能源部从后代<ol><li>选择母鹿被挤奶。里夫斯&#39;麂鹿一般一个小鹿2生出。牛奶中的母鹿分娩后的第二天，让小鹿机会获得初乳8获得必需的营养物质。 注：小鹿一般隐藏和proc中没有再次出现edure;母 - 子对的分离是没有必要的。 </li></ol>催产素，麻醉和眼部润滑剂2。管理<ol><li>辖10个国际单位/公斤催产素肌肉注射（IM）。催产素是一种激素，作用于女性哺乳期的乳腺，刺激乳汁的松懈下来。催产素可以从兽医收购。 </li><li>管理BAM麻醉IM 11布托啡诺含有0.45毫克/千克，二甲苯胺噻嗪0.25毫克/千克，medetomadine 0.07毫克/千克。在BAM麻醉剂混合物中的一种剂量镇静一个麂约20-30分钟，可提供充足的时间完成收奶。对麻醉剂的另一个选项是咪达唑仑在1-2毫克/千克，这将镇静一个麂为约10-15分钟。虽然这提供了足够的时间用于奶收集，这种麻醉剂被代谢的速度比BAM和动物可能试图逃脱在较短的时间周期的结束。 注：对疼痛的反应最小，在触摸耳朵较小的反射，其次是R1的持有MJ而不争的能力，表明该动物已成功麻醉。 注意：在一个麂没有充分与原剂量麻醉的情况下，研究者可以管理另一¼或麻醉剂混合物的原剂量取决于镇静的动物和/或电平的大小已经是的半实现的。但是，这可能增加不良反应麻醉的机会，并且还可能会导致麂更加深度麻醉和/或麻醉的时间较长。 </li><li>涂抹少量的润滑剂，以每只眼睛和蔓延的角落，以防止麂的眼睛在麻醉状态下干燥时。 </li></ol> 3，牛奶收集奶源是最容易与两位研究人员进行的;一位研究人员举行麻醉中麂的腿上，而手动表达奶（简称R1），第二研究员保持麂的头在一个舒适，安全的位置，并收集牛奶（R2）。 <ol><li>根据所使用的麻醉剂，挤奶应执行要么1）在他们的笔，他们应该在手术过程中醒来（咪达唑仑诱导）或2）的床上用品，免费接待室，以保持清洁（BAM - 提供一个安全的地方为他们诱导）。 </li><li> R1和R2应配备乙醇浸泡过的纱布和一些的15毫升锥形聚丙烯管来保存牛奶。 </li><li>由于催产素开始生效，放乳将成为麂的乳腺区可见。 R1：消毒戴手套的手和手指，以及整个乳房，并与乙醇浸泡过的纱布动物的每个单独的奶头。 R2：按住麂的头在一个安全和舒适的位置，使其很容易呼吸，并有助于防止瘤胃返流。按住收集管CL<html> OSE的乳头挤奶不接触，以防止潜在的污染。 </li><li>从一个乳头挤出乳汁通过使用拇指和食指轻轻挤压乳房组织中的向上运动，直至乳头充血与牛奶，并轻轻地滚动牛奶出奶嘴和成R2的保持管（ 见图2 ）。 </li><li>注意：牛奶出来，在一个流中，所以对于R 2具有收集管定位，使得很少或几乎没有牛奶将被浪费是重要的。 </li><li>本麂的乳房被分成象限。 R1：继续从每个象限挤出乳汁，直到牛奶不再轻易流动。如果牛奶不能从一个乳头表示，从所有其他乳头返回到第一前表达。它既是可以接受的，有用回来，并不断表达的牛奶来自同一个乳头是否应该产生比另一个更牛奶。 </li></ol>在母鹿4。麻醉逆转T“&gt;这是常见的咪达唑仑诱导母鹿开始在挤奶过程中醒来。在大多数情况下，美国能源部将试图逃避所有的牛奶已经表示之前。小的克制是用在努力增加牛奶产量，但无论是研究人员和美国能源部的安全性和舒适性也同样重要。 <ol><li>辖阿替美唑在2.5毫克/毫克medetomadine皮下注射BAM的快速逆转麻醉母鹿。 注意：使用这两种类型的麻醉剂，有必要保持密切的观察母鹿的，直到她能走在她自己没有绊倒或下降，并能吃喝不用担心误吸或窒息。没有不良影响已经注意到对于小鹿的收奶后，护士的能力。 </li></ol>

<ol>
	<li>Dueling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Muntiacus reevesi. Animal Diversity Web. , (2004).</li><li>Pei, K., Taber, R., O'Gara, B., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+cycle+of+the+Formosan+Reeves+muntjac+(Muntiacus+reevesi+micrurus)+in+northern+Taiwan,+Republic+of+China.">Breeding cycle of the Formosan Reeves muntjac (Muntiacus reevesi micrurus) in northern Taiwan, Republic of China.</a> Mammalia. 59 (2), 223-228 (1995).</li><li>Browning, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+prions+from+mule+deer+and+elk+with+chronic+wasting+disease+to+transgenic+mice+expressing+cervid+PrP.">Transmission of prions from mule deer and elk with chronic wasting disease to transgenic mice expressing cervid PrP.</a> J. Virol. 78 (23), 13345-13350 (2004).</li><li>Van de perre, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+antibody+via+mother's+milk.">Transfer of antibody via mother's milk.</a> Vaccine. 21 (24), 3374-3376 (2003).</li><li>Maden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+mice+produce+human+milk+protein.">Genetically modified mice produce human milk protein.</a> BioNews. , (2009).</li><li>Rodgers, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+milk+collection+in+the+rat.">Practical aspects of milk collection in the rat.</a> Lab. Anim. 29 (4), 450-455 (1995).</li><li>Depeters, E. J., Hovey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+collecting+milk+from+mice.">Methods for collecting milk from mice.</a> J. Mammary Gland Biol. Neoplasia. 14 (4), 397-400 (2009).</li><li>Knight, C. H., Maltz, E., Docherty, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+yield+and+composition+in+mice:+effects+of+litter+size+and+lactation.">Milk yield and composition in mice: effects of litter size and lactation.</a> 84 (1), 127-133 (1986).</li><li>Hedrich, H. J., Bullock, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+mouse.">The laboratory mouse.</a> , 735-736 (2004).</li><li>Nishimori, K., Young, L. J., Guo, Q., Wang, Z., Insel, T. R., Matzuk, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+is+required+for+nursing+but+is+not+essential+for+parturition+or+reproductive+behavior.">Oxytocin is required for nursing but is not essential for parturition or reproductive behavior.</a> Proc. Natl. Acad. Sci. USA. 93 (21), 11699-11704 (1996).</li><li>Miller, B. F., Osborn, D. A., Lance, W. R., Howze, M. M., Warren, R. J., Miller, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butorphanol-azaperone-medetomidine+for+immobilization+of+captive+white-tailed+deer.">Butorphanol-azaperone-medetomidine for immobilization of captive white-tailed deer.</a> 45 (2), 457-467 (2009).</li></ol>

The authors have nothing to disclose.

鼠标 有几个因素来自鼠标收集牛奶时要考虑到，包括1）的时间预留收集，催产素2）的给药剂量，3）有多少幼仔大坝是目前护理和4）金额自分娩已经通过在收集时的时间。使用以往的研究为指导，我们开始着手优化产奶量的条件。 此前有研究直接相关的产奶量与产仔8。不幸的是，产仔数是不能被控制的变量。然而，挤奶后的分娩?...

动物模型中提供的洞察，不能由体外分析来获得病的病理。提供最有效的结果，以使用密切相关的疾病和感兴趣物种的动物模型是重要的。例如，在穿过了麂（ 麂 ），小亚鹿1,2，和转基因小鼠中表达鹿科朊病毒蛋白（CerTgPrP）3的有用的动物模型鹿科物种。这两个物种是polyestrous，让常年繁殖，因此一致的来源与妊娠有关的组织和体液中，研究具体的机制在生物学鹿科。牛奶有研究的完成在动物模型中比在人类中应用较为简单（且便宜）繁多。研究人员可以调查乳和初乳为1）感染性疾病传播的潜在来源，2）在开发板的由母亲传递给后代的免疫球蛋白被动免疫4和3）乳铁蛋白pment，参与被动免疫的研究人员目前正在试图人类母乳中发现的一种蛋白质商业化生产5。 由小动物收集的牛奶大量可以证明是一项艰巨的任务。罗杰斯提出了一个方法，从大鼠6，随后在小鼠用于收集牛奶。 DePeters和霍维提出了两种方法，奶收集，一种使用由连接到巴斯德吸管的橡胶吸管灯泡产生的手动产生的真空，并且第二要求挤奶单元，然后将其连接到一个真空源（建造这样作为一个水龙头）收获鼠标奶7。在这里，我们提出了一个简单的，低成本的方法从小鼠和里夫斯&#39;麂鹿，只需要现成的实验室设备和基本的技术技能收集牛奶。我们的方法产生足量的牛奶各种应用程序。

<table border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="28">
			<td height="28" style="height:28px;width:360px;">Materials-Mouse</td>
			<td style="width:297px;"></td>
			<td style="width:236px;"></td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Reagent</td>
			<td>Vendor</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine (KetaVed 100 mg/mL) (dose: 80-100 mg/kg)</td>
			<td>MWI/Vedco</td>
			<td>011075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (TranquiVed 100 mg/ml) (dose:5-10 mg/kg)</td>
			<td>MWI/Vedco</td>
			<td>NADA 139236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye lubricant</td>
			<td>MWI/Apexa</td>
			<td>701013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (2 IU/kg)</td>
			<td>Vet One</td>
			<td>501013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">Materials- Muntjac</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Reagent</td>
			<td>Vendor</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Midazolam (1-2 mg/kg)</td>
			<td>Amerisource Bergen Drug Corp</td>
			<td>924326</td>
			<td>optional: researcher can either use Midazolam on its own or BAM combination as an anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butorphanol tartrate (Torbugesic) (0.45 mg/kg)</td>
			<td>Fort Dodge Animal Health</td>
			<td>NADA 135-780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Azaperone tartrate (0.25 mg/kg)</td>
			<td>ZooPharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medetomadine HCl (0.07 mg/kg)</td>
			<td>ZooPharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antipamezol hydrochloride (Antisedan) (dose: 0.25 mg/kg)</td>
			<td>Orion Corporation</td>
			<td>NADA 141-033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (10 IU/kg)</td>
			<td>Vet One</td>
			<td>501013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">Equipment- Mouse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Equipment</td>
			<td>Vendor</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P-200 Pipetman</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#956;L pipette tips</td>
			<td>Light Labs</td>
			<td>B-2004-SH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td>MWI</td>
			<td>005427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">27 gauge needles</td>
			<td>MWI</td>
			<td>002031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">Equipment- Muntjac</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Equipment</td>
			<td>Vendor</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml conical tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22 gauge needles</td>
			<td>MWI</td>
			<td>873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 ml luer lock syringes</td>
			<td>MWI</td>
			<td>001377</td>
			<td></td>
		</tr>
	</tbody>
</table>

milk collection methods for mice and reeves' muntjac deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

鼠标 从我们的实验中，我们已经确定，就可以收集大约100-400微升的牛奶从一个实验室小鼠，取决于几个变量。这些变量包括：1）的时间预留收集，2）催产素的剂量给药，3）有多少幼仔大坝是目前护理和时间分娩后4）的金额。我们的研究表明，当坝从它们的后代中分离至少2小时，获得最高的产率，和至少45分钟到一个小时被设定每只小鼠挤奶一边。最大的牛奶...

Watch this Scientific Journal Video about Milk Collection Methods for Mice and Reeves' Muntjac Deer at JoVE.com

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

牛奶收集方法小鼠和里夫斯&#39;麂鹿

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

milk-collection-methods-for-mice-and-reeves-muntjac-deer

Research

JoVE Journal

Biology

Milk Collection Methods for Mice and Reeves' Muntjac Deer

19.7K 次观看.  Colorado State University. Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves’ muntjac deer. 

视频: Milk Collection Methods for Mice and Reeves' Muntjac Deer

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae子囊孢子生产和收集微阵列实验。

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

科研

生物学

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

海水采样和收集

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

剖检诊断，并选择在大鼠和小鼠的组织和样品收集

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

奶源在大鼠毛细管管和乳脂肪含量Creamatocrit估计

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

测定野生型肝脏和牛奶样品中的唾液酸<em&gt; CMAH</em&gt;敲出小鼠

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

阿斯蒂亚纳斯墨西哥的Gamete收集与体外受精

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

蝙蝠组织收集 -细胞分析和原细胞培养

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...