这项研究是由加拿大自然科学和工程研究理事会（NSERC，401848-2011到MP）的支持。 MP从全宗德RECHERCHE魁北克-桑特（FRQ-S）获得了职业生涯奖。 LE是从FRQ-S（FRQ-S 14D 29441）的博士研究金的接收者。我们感谢玛丽 - Josée沃尔曼技术援助。

伦理声明:所有涉及动物的协议是由科米特去保护DES ANIMAUX DE L&#39;拉瓦尔大学批准，并按照加拿大动物保护协会的指南实验动物的护理和使用（编2）被做了。这里所述的方案对于大约800克成年动物优化。固定的量应根据动物的大小进行调整。 1.一种用于Transcardiac灌注溶液的制备<ol><li>根据以下步骤制备1升50mM的磷酸钠的磷酸盐缓冲盐水（PBS）溶液。灌注之前制备至多24小时的溶液，并保持在4℃直至使用。 <ol><li>测量800毫升蒸馏水在1升烧杯中。添加5.87克磷酸氢钙无水的钠（Na 2 HPO 4）中加入1.20g磷酸二氢钠一水合物的（的NaH 2 PO 4·H 2 O），和9g的氯化钠（NaCl）。搅拌溶解。 </li><li>通过逐渐加入5 N氢氧化钠调节pH至7.4。添加蒸馏水达到100 L的总体积 注意:将NaOH是腐蚀性化学化合物。穿相应的个人防护设备（PPE;实验室外套，手套，护目镜等 ）。 </li></ol></li><li>制备2升根据下面的步骤4％低聚甲醛（PFA）的。该解决方案应准备手术前至多24个小时，并保持在4℃直至使用。 <ol><li>测量1.5升的蒸馏水中的2升烧杯中。添加23.48克二元无水磷酸的钠（Na 2 HPO 4）和4.80克磷酸二氢钠一水合物的（的NaH 2 PO 4·H 2 O）。搅拌溶解。添加蒸馏水达到200 L的总体积</li><li> 55℃ - 直至温度达到约45加热排气罩下的溶液中。不要加热到60℃以上。 </li><li>逐渐80克PFA的添加到该溶液中并搅拌直到完全溶解（约30 - 60分钟）。保持监测温度以保持其在60℃以下。 注意:PFA处于其粉末形式高挥发性。这是如果有剧毒的眼睛或皮肤接触，并在吸入或食入的情况下危险。穿PPE和排气罩下谨慎使用。 </li><li>冷却该溶液至4℃，并过滤。储存于4℃。 </li></ol></li><li>根据以下步骤在0.1M磷酸盐缓冲液（PB）制备1升3％的丙烯醛。该PB溶液应该至多24个小时灌注之前被制备。 注意:丙烯醛是如果吸入，并且能够产生直接的伤害具有高毒性。这也是腐蚀性和剧毒，如果通过皮肤吸收。它是致癌和致突变性。穿相应的PPE和通气罩下使用。 <ol><li>测量800毫升蒸馏水。添加8.66克磷酸氢钙无水硫酸钠（ 硫酸钠 </子&gt; HPO 4）和5.38克磷酸二氢钠一水合物的（的NaH 2 PO 4·H 2 O）。搅拌，直到所有的盐溶解。加蒸馏水至达到1 L.的总体积保持在4℃下该溶液中。 </li><li>手术前，转移900毫升的PB溶液到1L的玻璃容器中，并添加30.94毫升的97％的丙烯醛溶液通气罩下。添加PB溶液达到1升，搅拌的总体积。过滤溶液，并保持它在4℃下。 </li></ol></li></ol> 2. Transcardiac灌注和脑解剖<ol><li>在整个手术过程中保持在冰上的解决方案。 （50毫PBS）和上直到没有空气保留在软管将泵通过将管在所述第一溶液中使用制备泵。对于猕猴的transcardiac灌注，使用21号针，并设置流出在大约80毫升/分钟。 </li><li>麻醉动物提供一个组合的一个肌内注射氯胺酮（20毫克/千克），甲苯噻嗪（4毫克/千克），并乙酰丙嗪（0.5毫克/千克）的真实存在。保持下异氟烷（3％）镇静动物。 </li><li>附上动物的四肢通风表。 </li><li>用手术刀，去除皮肤到腋窝。切开腹部肌肉。使用重型手术剪刀小心地避免重要器官横向切割的肋骨。 </li><li>切手术剪隔膜，提高肋骨暴露心脏。一旦隔膜被切断，迅速地进行，因为心脏将停止分钟内跳动。 </li><li>删除用手术刀刀片心包和针头插入左心室。用手术刀，认真做一个小切除至右心房。在72毫升/分钟快速启动泵并保持针就位。如果可能的话，倾斜的动物才能拥有它的头比心脏较低的水平。 注意:要小心，不要插入针时刺穿室间隔。 <ol><li> RINSe为约300毫升的PBS直到肺血是白色的，没有血液出来的右心房。 </li></ol></li><li>停泵，快速软管/管转移到3％丙烯醛的解决方案，并重新启动水泵。一旦心脏已经停止了跳动逐渐提高抽速，以80毫升/分钟。灌注约500 mL丙烯醛溶液。 </li><li>停泵，快速软管转移到4％PFA解决方案，并重新启动水泵。该步骤需要约1升的PFA。 注:固定完成后，前肢是刚性和颈部僵硬。 </li><li>切头，仔细解剖出脑颅骨。注意不要用手术器械损伤大脑。灌注是最佳时的脑苍白（无血液曲线）和刚性（ 图1A）。 </li><li>浸入完整的脑在4％PFA中于4℃下1个小时。 </li><li>连续切割大脑与冷却振动切片机（4℃）在所期望的平面成50个微米厚的切片并收集它们在PBS（0.1M）（ 图1B）。 注意:此步骤可以以多种方式根据期望的协议来进行。半球可以事先分开，以振动切片机切割，或保持整体。如果大脑的振动切片机平台太大，它可以被切割成更小的块。此步骤之后，部分可被存储的时间在-30℃下在由40％的PB（50毫摩尔），30％甘油和30％乙二醇的防冻液长的时间。 </li></ol> 3.预嵌入免疫组织化学（图1C） <ol><li>准备如下的Tris缓冲盐水的4升储备溶液（TBS，50mM的，pH值7.6）。 <ol><li>测量2L蒸馏水在一个4升烧杯中，加入24.23克三羟甲氨基甲烷的（THAM; C 4 H 11 NO 3），并搅拌至溶解。 </li><li>调节pH至7.6用约148毫升的1N HCl。酸应该CAUTI被添加上避免达到低于7.6的pH值。的总体积应为4 L. 注意:盐酸是高度腐蚀性的。穿戴合适的个人防护装备。 </li></ol></li><li>含有感兴趣的区域选择部（来自步骤2.11），以用于免疫组织化学EM被处理。 </li><li>在室温洗PBS自由浮动部3X（0.1M，pH 7.4）中5分钟以冲洗防冻溶液。 </li><li>制备的NaBH 4 0.5％溶液在PBS中稀释。 <ol><li>权衡0.05克的NaBH 4和稀释在10mL PBS中。不包括。使用前准备这个解决方案。 </li></ol></li><li>在孵育在室温30分钟的新鲜制备的NaBH 4溶液中的部分。轻轻摇动。不包括。 </li><li>洗涤3次在PBS中持续10分钟，在室温，剧烈摇动，直到没有反应气体的保持。 </li><li>准备EM用2％适当正常血清和PBS中稀释的0.5％冷的鱼明胶封闭溶液。 注:量建议立即进行删除D IN为了计算有足够的用于以下三个步骤。使用从托管的二级抗体的同种动物做了血清。避免使用的抗原修复方法或添加少量的Triton（增加抗体的穿透），因为这些方法显著损害组织的质量。 </li><li>在孵育在室温下1个小时的封闭溶液的章节。轻轻摇动。 </li><li>准备在封闭溶液稀释的初级抗体溶液。 注意:第一抗体的浓度通常是相同LM免疫组织化学，但考虑与不同的抗体浓度进行测试预先，因为一些初级抗体可能不与丙烯醛工作。如果是这样，可以使用戊二醛的混合物（0.1 - 2％）和4％PFA中transcardiac灌注并获得类似的结果。优化培养时间，培养温度为好。 </li><li>孵育在初级抗体溶液中的部分隔夜在室温下用温和的摇摆。使用之前测试的孵育时间和温度。 </li><li>洗部3X的PBS在RT 5分钟轻微摇动。 </li><li>制备1:在封闭溶液稀释生物素化的第二抗体的溶液1000。 注:第二抗体必须针对用于产生第一抗体的宿主物种得到提升。 </li><li>孵育在第二抗体溶液中的部分在RT下1.5小时。轻轻摇动。 </li><li>制备二级抗体孵育结束之前至少60分钟的抗生物素蛋白 - 生物素 - 过氧化物酶（ABC）溶液。 <ol><li>使用校准的移液管来测量8.80μL/ mL的溶液A和B的和稀它们在PBS中。 </li><li>温和摇动在室温至少60分钟，以允许完全的抗生物素蛋白和生物素分子之间的结合。 </li></ol></li><li>在次级抗体溶液孵育后，洗涤3次在PBS中在RT下10分钟。 </li><li>孵育节在ABC这样lution在RT 1个小时。轻轻摇动。 </li><li>在TBS中洗一次在PBS中和两次在室温10分钟，轻轻摇动。 </li><li>制备0.05％3,3&#39;-二氨基联苯胺（DAB）的用0.005％H 2 O 2的TBS稀释新鲜溶液。 <ol><li>称取12.5毫克DAB的和稀释在25mL冷的TBS。避光。 注意:如果吸入民建联粉挥发性高，有害的。它是致癌和致畸。因此，当稀释时怀孕或哺乳妇女不宜操作这个产品，甚至。操作时，请使用N95口罩和穿PPE。 </li><li>过滤溶液，并在即将使用前添加30％H 2 O 2 4.5μL。 </li></ol></li><li>在孵育在室温下将DAB溶液3-7分钟的部分。轻轻摇动。 注:棕色沉淀不应该为了避免背景染色高水平太暗。培养时间应相应优化。 </li><li>通过快速洗涤两次停止反应在寒冷的TBS，然后两次在RT冷TBS 10分钟，接着在PB为两次10分钟，用温和的摇动。 注意:使用PB（而不是PBS）的是为了消除的NaCl任何痕迹，因为它会与锇和形成晶体的反应是至关重要的。 </li></ol> 4. Osmification和嵌入的电镜观察<ol><li>制备1％的四氧化锇在PB稀释的溶液（OSO 4）。避光。 注意:锇是剧毒，不得与皮肤，眼睛或口腔接触，并且不应该被吸入。如果误食可导致死亡。它应该只在一个排气罩，并用适当的PPE使用。 </li><li>孵育在所述排气罩（不搅拌）下在室温下30分钟的OsO 4溶液中的部分，并覆盖它们用铝箔以保护它们免受光。完全平坦化段之前将所述的OsO 4溶液。 注:部分变得非常黑暗和RIGID，应小心下面这个步骤进行操作。 </li><li>在osmification期间准备憎水环氧树脂。 <ol><li>添加环氧树脂混合物（20克环氧树脂，20g的硬化剂，0.6g的加速器和0.4g增塑剂）的各成分的合适的量为大塑料杯中。用木棒或塑料移液管搅拌，直到获得均匀的棕色。 注:这是利用各组分的准确比例的关键。 </li><li>转移等量至适当大小的铝杯，根据段的数量以进行处理。让它休息。 </li></ol></li><li>洗osmificated切片在PB 3倍，在室温10分钟以低速摇摆。 </li><li>脱水在以下系列梯度的乙醇的部分为每次2分钟:2次，35％的乙醇;每个在50，70，80，90，和95％乙醇1周时间;和3X在100％乙醇。 </li><li>转移部分到玻璃瓶完成dehydrati上通过孵育切片3次，在环氧丙烷2分钟的过程。 注意:环氧丙烷是高挥发性和毒性有机溶剂。它可引起接触的情况下，或者如果吸入或摄入眼睛或皮肤造成严重损害。它已被列为2级致癌物质。它应该只在一个排气罩和与PPE一起使用。这也是高度易燃，应远离任何热源。 注意:在这个步骤之前，部分应仔细在玻璃小瓶中转移，因为氧化丙烯是有机溶剂，并与塑料不相容。在这一点上，部分是很脆弱的，应小心进行操作。部分可以可选地转移至步骤4.5之前玻璃小瓶中。 </li><li>小心转移部分，一个接一个，在铝杯和避免与空气尽可能接触。平嵌入先前混合防水性环氧树脂的部分和VENTIN下过夜孵育他们在RT克罩。 注:在此步骤中，部分是完全脱水和非常脆弱，应小心进行操作。 </li><li>使用矿物油，制备润滑脂涂覆的玻璃载片与脂塑料盖玻片一起。 </li><li>至多在60℃温育铝杯12-15分钟软化树脂。小心弄平在载玻片的脂侧的部分。放置润滑盖玻片小心推出任何残留的空气。 </li><li>孵育载玻片在60℃下48小时。 注:不超过48小时的培养时间，因为树脂将变得太硬，这一点至关重要。 </li><li>除去塑料盖玻片。 </li></ol> 5.样品制备超薄切片和观察用透射电子显微镜<ol><li>用望远镜找到感兴趣的区域，并切一小四片约1mm 2用手术刀。 </li><li>文件的树脂块的前端与胶曲adrangular片在其上（ 图1C）。让胶水干燥至少1小时之前或切片过夜。 </li><li>使用超薄切片机，切四边形片成80微米厚的切片（ 图1D）。 <ol><li>代替树脂块在垂直位置超薄切片甲板和，用锋利的剃刀刀片，逐渐切断树脂块的每一侧，以形成具有光滑侧面的梯形。 </li><li>把甲板在其水平位置和旋转，直到梯形的最长边朝下的块。 </li><li>使用金刚石修整工具或玻璃刀的四边形片的表面修整。设置超薄切割300个微米厚的切片以1毫米/秒。调整刀能够上下平行四边形片并显示大约1°非常小的水平角度。 注意:该角度将允许用户以接近组织几乎平行于B的表面锁定，其中免疫标记元素更容易被发现。当使用这些参数与金刚石修整工具，该树脂显示有光泽的白色。当组织被切断，它将变为紫色或绿色的颜色。 </li><li>使用配备有装有蒸馏水船切为80μm厚的切片的超45°的金刚石刀，通过使在它们用一块吸收二甲苯放倒纸的平滑部分，并且收集上涂布聚乙烯醇缩甲醛 - 镍槽栅格或连续切片裸150个目铜网上（ 图1E）。 </li></ol></li><li>将网格的网格储物盒。 </li><li>染色柠檬酸铅的网格。 <ol><li>使用5毫升的注射器和一个0.2微米的针筒式过滤器，以制备1:过滤柠檬酸铅储备溶液1溶液并过滤蒸馏水。避光保存它。 注:柠檬酸铅的储备液应新鲜每月以避免固体depos形成它的。请参阅库存配方中的材料数据表。此外，如果多个系列电网需要进行染色，当它变成乳白色改变稀释液。 </li><li>每个网格放置到稀释的溶液的液滴，在与溶液接触的部分。孵育3分钟。 </li><li>用小镊子举行的网格，并彻底冲洗含蒸馏水两个烧杯。 </li><li>轻轻地用吸油纸去除多余的水。存储网格的网格框。等待检查由透射电子显微镜的截面前30分钟（TEM; 图1F）。 </li></ol></li></ol>

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在本文中，我们提出了非人类的灵长类动物和适于EM样品检查嵌入预免疫的transcardiac灌注一个可靠的协议。虽然典型的冷冻电镜，如CEMOVIS，提供脑组织超微结构的保存较好，这也限制了使用免疫组织化学12。其它技术，包括冷冻替代和Tokuyaso技术中，允许后嵌入免疫组织化学，但是这些技术是昂贵的，由于该过程期间所需的附加设备，并且可以是费时和技能挑战<...

光镜，包括共焦和双光子显微镜，已被证明是用于体内神经元过程研究，除其他事项外1,2的有效工具。虽然在光镜（LM）级的典型的空间分辨率约为200nm时，使用不同的光源，如极紫外和软X射线显微术最近的技术进步，已经此分辨率显着增加至近10纳米的空间分辨率3 ，4，5。在其他成像技术的进步包括结合组织学磁共振成像和用于测量体内髓鞘，只在6电子显微镜（EM）的水平，7是传统上可测量的参数的厚度提供一种新颖的方法。 AlthouGH这些进步在LM水平用于研究活的过程，结构的详细视图和表征，如突触联系提供一个极好的工具，只能与EM，它提供的分辨率，可以达到0.5nm的来实现。然而，观察在EM级要求的标本是死的，并改变在某些方面，化学固定剂和脱水过程中，为了保持细胞结构。因此，在高分辨率检验的生物样品可以是具有挑战性由于从电子束辐射损伤，对比度低，膜的结构偏差，或可能出现以下脱水和环氧树脂包埋8，9，10的伪像甚至存在。 在用于结构分析它们的天然形式保存标本可以通过使用"EM冷冻玻化部分的"或CEMOVIS，一个切片的方法来实现该涉及快速冷冻和嵌入在玻璃体冰样品和在低温下11,12检查EM下的部分。这一程序可以检测样本的检验，而他们仍然坚实，充分水化，从而消除由于脱水处理的工件13。然而，该方法涉及为低温超薄切片额外的设备，以及在标准EM附加的设备，以允许该观察在非常低的温度，这产生显著额外的成本。此外，CEMOVIS方法排除了使用免疫标记技术中，由于抗体通常具有在RT下温育的。可替代地，有可能通过使用冷冻替代方法中，在此期间，而在低温保护化学品和浸入第是低温固定试样缓慢解冻与免疫组织化学程序超微结构分析结合恩嵌入专用树脂，如Lowicryls。后嵌入免疫标记然后可以在这样的材料12进行。然而，冷冻取代和冷冻固定技术是耗时的。它们需要额外的设备的安装，但是仍然需要样品被暴露于有机溶剂和化学固定剂可改变细胞结构，尽管使用一个低温14,15。因此，尽管两者在LM和EM水平，脑组织的化学固定，特别是丙烯醛的所有技术的进步，仍然是一个低成本的和时间有效的方法来免疫组织化学EM 16结合起来。 在过去的几十年，进行了许多尝试，发现提供最佳的组织保存醛的混合物。 20世纪60年代之前，这给了可接受的结果为EM的唯一化学固定是四氧化锇。然而，四氧化锇是剧毒和昂贵的，通过血管系统以固定的器官如脑排除其使用。丙烯醛在50年代后期推出了作为适用于蜂窝结构17的EM观察动物组织保存的可靠方法。它更深入地穿透组织并且比通过浸渍用于固定时，并且允许细胞质成分的良好的保存，与组织17的最小的收缩其它醛反应更迅速。在新鲜组织使用时，通过使活分子化合物，如酶和其它蛋白质18的更精确的定位，这种特征给丙烯醛固定比其它醛明显的优势。事实上，它已经经过多年用于可视化在许多物种，包括两栖类和啮齿类动物的EM水平固定的一种简单，高效，低成本的方法验证，因为它有效地stabiliZES肽和蛋白质，保留抗原性，并与另一种醛组合使用固定剂16，18，19，20，21时提供了相对完整超微结构。用于在啮齿动物丙烯醛固定协议已自然后被标准化并且广泛地使用，特别是由皮克尔组，以实现双重免疫标记为EM 16，22。几组在非人类灵长类动物的大脑组织23已经使用丙烯醛固定。然而，据我们所知，有效率地描述了在非人类的灵长类丙烯醛是与EM免疫标记24兼容的化学固定只有一个出版协议。 在本文中，我们提供了一种简单而可靠的方法以有效地化学修复非哼声一个灵长类动物大脑中的丙烯醛，允许预先埋设 - 免疫标记和传输EM检查沿着一个潜在的长期保存。

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Dibasic anhydrous sodium phosphate (Na2HPO4)</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">S374-500</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">Monobasic monohydrate sodium phosphate (NaH2PO4.H2O)</td>
			<td style="width:123px;">EM Science</td>
			<td style="width:119px;">SX0710-1</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride (NaCl)</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">S271-3</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydroxymethyl aminomethane (THAM)</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">T370-500</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">HCl&#160;</td>
			<td style="width:123px;">EMD</td>
			<td style="width:119px;">HX0603-3</td>
			<td style="width:177px;">1N dilution. Product is corrosive. Use with appropriate potection.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NaOH</td>
			<td style="width:123px;">EMD</td>
			<td style="width:119px;">SX0590-1</td>
			<td style="width:177px;">5N dilution. Product is corrosive. Use with appropriate protection.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paraformaldehyde</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:177px;">4% dilution. Product is highly volatile in its powder form and highly toxic. Use with caution under a venting hood with appropriate protection.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Acrolein (90%)</td>
			<td style="width:123px;">sigma</td>
			<td align="right" style="width:119px;">110221</td>
			<td style="width:177px;">3% dilution. Product is highly toxic. Use under a venting hood with appropriate protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autopsy venting table</td>
			<td style="width:123px;">Mopec</td>
			<td style="width:119px;">CE400</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Electronic perfusion pump</td>
			<td style="width:123px;">cole parmer</td>
			<td style="width:119px;">masterflex L/S 7523-90</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle (perfusion)</td>
			<td style="width:123px;">terumo&#160;</td>
			<td style="width:119px;">NN-1838R</td>
			<td style="width:177px;">18G 1 1/2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle</td>
			<td style="width:123px;">terumo&#160;</td>
			<td style="width:119px;">NN-2713R</td>
			<td style="width:177px;">21G 1/2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;">20 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;">4 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;">0.5 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scalpel blades</td>
			<td style="width:123px;">Feather&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; lance</td>
			<td style="width:119px;">201011&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; J9913</td>
			<td style="width:177px;">No.22 for surgery and&#160;&#160;&#160; No. 11 for EM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical scissors</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rongeurs</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vibratome</td>
			<td style="width:123px;">Leica</td>
			<td style="width:119px;">VT 1200S</td>
			<td style="width:177px;">Calibrate blade before each use, when the device allows it</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome razor blade</td>
			<td style="width:123px;">Gillette</td>
			<td style="width:119px;">GIN 642107</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">G33-4</td>
			<td style="width:177px;">30% dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">E178-4</td>
			<td style="width:177px;">30% dilution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Sodium borohydride (NaBH4)</td>
			<td style="width:123px;">sigma</td>
			<td style="width:119px;">S-9125</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Normal horse serum</td>
			<td style="width:123px;">Jackson immunoResearch Laboratories</td>
			<td>008-000-121</td>
			<td style="width:177px;">2% dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cold-fish gelatin</td>
			<td style="width:123px;">Aurion</td>
			<td style="width:119px;">900.033</td>
			<td style="width:177px;">0.5% dilution. Original product is concentrated at 40%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Primary antibody, SERT</td>
			<td style="width:123px;">Santa Cruz biotechnology</td>
			<td style="width:119px;">SC-1458</td>
			<td style="width:177px;">1/500 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Primary antibody, ChAT</td>
			<td style="width:123px;">Chemicon (Millipore)</td>
			<td style="width:119px;">AB144P</td>
			<td style="width:177px;">1/25 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary antibody, TH</td>
			<td style="width:123px;">ImmunoStar</td>
			<td style="width:119px;">22941</td>
			<td style="width:177px;">1/1000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated secondary antibody, goat</td>
			<td style="width:123px;">Vector laboratories</td>
			<td style="width:119px;">BA-9500</td>
			<td style="width:177px;">1/1000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated secondary antibody, mouse</td>
			<td style="width:123px;">Vector laboratories</td>
			<td style="width:119px;">BA-2000</td>
			<td style="width:177px;">1/1000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectastain elite ABC kit</td>
			<td style="width:123px;">Vector laboratories</td>
			<td style="width:119px;">PK6100</td>
			<td style="width:177px;">8.8 &#181;L/mL of A and B each</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">3&#39;3 diaminobenzidine (DAB)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">D5637</td>
			<td style="width:177px;">0,05% dilution. Product is highly volatile in its powder form and toxic. Do not throw waste in the sink.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Peroxide (H2O2) 30%</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">H-323</td>
			<td style="width:177px;">0,005% dilution</td>
		</tr>
		<tr height="212">
			<td height="212" style="height:212px;width:216px;">Osmium tetroxide (OsO4)</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">2% 19152&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 4% 19150&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:177px;">Original solution can be either 2% ou 4%. Keep attention to which one is used to calculate the final 1% dilution. Product is very sensitive to light. Osmium is highly toxic. Use only under a venting hood with appropriate protection.</td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:216px;">Durcupan water-repellent epoxy resin</td>
			<td style="width:123px;">sigma</td>
			<td style="width:119px;">A: M epoxy resin (44611)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; B: hardener 964 (44612)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; C: accelerator 960 (DY 060)&#160; (44613)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; D: plasticizer (44614)&#160;</td>
			<td style="width:177px;">Polymerize 48h at 58 &#176;C before throwing in waste.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Alumium cups</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">70048-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethanol</td>
			<td style="width:123px;">commercial alcohols</td>
			<td style="width:119px;">1019C</td>
			<td style="width:177px;">Dilute in distilled water with appropriate concentration</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Propylene oxide</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">20401</td>
			<td style="width:177px;">Organic solvent. Highly volatile and toxic. Use under a venting hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-coated medium glass slides</td>
			<td style="width:123px;">brain research laboratories</td>
			<td style="width:119px;">3875-FR</td>
			<td style="width:177px;">Grease surface with mineral oil</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Plastic film (Aclar embedding film)</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">50425-25</td>
			<td style="width:177px;">Grease surface with mineral oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultramicrotome</td>
			<td style="width:123px;">Leica UC7</td>
			<td style="width:119px;">EM UC7</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Diamond trimming tool (ultratrim)</td>
			<td style="width:123px;">Diatome&#160;</td>
			<td style="width:119px;">UT 1081</td>
			<td style="width:177px;">Can use glass knife alternatively</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra 45&#176; Diatome Diamond knife</td>
			<td style="width:123px;">Diatome&#160;</td>
			<td style="width:119px;">MC13437</td>
			<td style="width:177px;">equipped with a boat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylenes</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">X5SK-4</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">150-mesh copper grids</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">G150-cu</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">grid-box</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">71138</td>
			<td style="width:177px;">Can store up to 100 grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium citrate</td>
			<td style="width:123px;">Anachemia</td>
			<td style="width:119px;">81983</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="336">
			<td height="336" style="height:336px;width:216px;">Lead nitrate</td>
			<td style="width:123px;">sigma</td>
			<td style="width:119px;">L-6258</td>
			<td style="width:177px;">Make a stock solution of lead citrate made of 1.33 g of lead nitrate and 1.76g of sodium citrate diluted in 42 mL of pre-boiled and cooled distilled water to which 8 mL of 1N NaOH are added after the conversion from lead nitrate to lead citrate is complete. pH should be approximately 12. Store solution in a hermetic plastic bottle and protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe</td>
			<td style="width:123px;">terumo&#160;</td>
			<td style="width:119px;">SS-05L</td>
			<td style="width:177px;">5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe filter</td>
			<td style="width:123px;">corning</td>
			<td style="width:119px;">431222</td>
			<td style="width:177px;">0.2 &#181;m</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Absorbing paper (bibulous paper)</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">70086-1</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:123px;">Laboratory film</td>
			<td style="width:119px;">PM-999</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mineral oil</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">M5904</td>
			<td style="width:177px;"></td>
		</tr>
	</tbody>
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preparation of non-human primate brain tissue for pre-embedding immunohistochemistry and electron microscopy

尽管在光学显微镜级别的所有技术的进步，电子显微镜仍然是神经科学的唯一工具来检查和鉴定神经元，如突触联系的超微结构和形态的细节。脑组织的保存好电子显微镜可以通过严格的冷冻固定的方法来获得，但这些技术是相当昂贵和限制使用免疫标记，这关键是要了解确定的神经系统的连通性。冷冻替代方法已经被开发，使冷冻固定的与免疫标记的组合。然而，这些方式方法的重现性通常依赖于昂贵的冷冻设备。此外，获得可靠的结果，这种技术是非常耗时和技能挑战。因此，传统的化学固定脑，特别是丙烯醛的固定剂，仍是电子结合了时间效率和低成本的方法显微镜用免疫组化。在这里，我们提供使用化学丙烯醛固定，导致灵长类动物脑组织的保存，并与嵌入前免疫电镜和透射电镜检查相适应的可靠的实验方案。

在本节中，我们提出获得，继观察，在发送EM水平，进行免疫染色的灵长类动物的脑组织化学固定用3％丙烯醛的混合物和4％PFA的代表性结果。我们所取得的超微结构的良好保存，由相对完整髓鞘和双膜（ 图2A）的可视整齐所示。突触联系，从微环境的神经元元件，均可以很容易被识别（ 图2B）。标记的二氨基联苯胺（DAB）免疫沉淀物的神经元元?...

Watch this Scientific Journal Video about Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy at JoVE.com

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

在这里，我们提供了一种简单，低成本，和时间高效的协议进行化学固定与丙烯醛固定液灵长类脑组织，从而允许长期保存，其与预嵌入-免疫组化透射电子显微镜兼容。

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

preparation-non-human-primate-brain-tissue-for-pre-embedding

Research

JoVE Journal

Neuroscience

14.0K 次观看.  Université Laval. 在这里，我们提供了一种简单，低成本，和时间高效的协议进行化学固定与丙烯醛固定液灵长类脑组织，从而允许长期保存，其与预嵌入-免疫组化透射电子显微镜兼容。 

视频: Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

制备小鼠脑组织中的免疫电镜

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

科研

神经科学

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

为揭示复杂的大脑地形wholemount免疫组化

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

引物对免疫冰冻切片大鼠脑组织:例为染色小胶质细胞和神经元

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

使用串行块面部扫描电子显微镜脑线粒体分析

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

在麻醉和清醒行为的非人灵长类动物大脑中注射用于光遗传学的AAV载体

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

用于登德里脊柱研究的串行块面扫描电子显微镜 （SBEM）

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...