We thank the Pathology Research Core for assistance with electron microscope analysis studies at Cincinnati Children's Hospital Medical Center. The work was supported by NIH (American Society of Hematology Bridge award to-MDF; R01 DK102890 to MDF).

所有试验程序都通过在辛辛那提儿童研究基金会的体制动物委员会批准。 4个月 - 在本研究中，造血干细胞从C57BL / 6年龄2近交系小鼠的骨髓中分离。细胞使用具有不同表面标志包括世系，的c-Kit，的Sca-1的BM染色后的FACS分选，Flt3的和CD34用于基于Lin-的的Sca-1 +的c-Kit +的Flt3的HSC纯化- CD34 -门控策略作为标准协议8如前所述。 注意:一些剧毒化学品在此过程中使用。这些是吸入和皮肤接触有毒。请戴手套和防护服。在通风橱中使用这些化学品工作时的工作。 1.细胞，固定，染色和预嵌入<ol><li>排序万的造血干细胞（使用林-的Sca-1 <suP&gt; +的c-Kit +的Flt3 - CD34 -门控策略; LT-HSC种群）中含有1.5 ml离心管600微升的Iscove氏改良的Dulbecco氏培养基（IMDM）与使用FACS 10％胎牛血清。 </li><li>降速排序的造血干细胞在1000 XG使用的离心分离机摇出水桶在1.5 ml离心管10分钟。通过温和抽吸除去培养基，并在每个步骤中的管离开200微升培养基。在这个阶段，将细胞沉淀保留在管不可见的。 </li><li>通过加入0.2ml在室温（RT）下2个固定剂溶液1小时1,9固定细胞。 <ol><li>使用前固定液新鲜。在1倍，将溶液在0.1M二甲胂缓冲器包含3％多聚甲醛和2.5％戊二醛。 </li></ol></li><li>离心机使用具有开式水桶离心机将细胞在30℃1000×g离心10分钟。 </li><li>洗涤细胞用600微升0.1M的二甲胂缓冲采用离心法和离心后留下200微升缓冲在管。 </li><li>通过在二甲胂酸盐缓冲液中加入200微升的伊文思蓝的2毫克/毫升溶液染色固定的细胞和在室温下孵育20分钟。这些细胞会被蓝色。 </li><li>洗涤细胞3次用600微升的0.1M二甲胂酸盐缓冲液使用离心，每次离开200μl的缓冲液在管中。 </li><li>重悬在200μl缓冲液中的细胞和细胞悬浮液转移到0.5毫升管。离心机在1000×g离心用台式离心机10分钟。删除缓冲轻轻地，而不会干扰现在可见的微小细胞沉淀，并留下50微升的缓冲区管。 </li><li>琼脂糖加入200毫克低熔点到5ml的0.1M二甲胂缓冲剂在15ml试管为4％的琼脂糖溶液。通过在100毫升玻璃瓶中的管传送到沸水熔化琼脂糖和直至使用保持冷淡。 </li><li>加入200微升4％低熔点的琼脂糖溶于0.1M cacodylaTE缓冲液以在0.5ml管中的细胞悬浮液，并立即用台式离心机离心在1000 xg离心10分钟，在30℃。 </li><li>确认细胞沉淀在0.5毫升管的底部进入离心后半固体琼脂糖之后，立即转移管至4℃或冰上20分钟，以固化琼脂糖。这一步骤是非常关键的，因为在细胞的电子显微镜下的好簇此步骤的结果的小可见细胞团块。 </li><li>小心含有从管的细胞沉淀凝固琼脂糖转移到用27克针3,10含有缓冲器35毫米塑料培养皿中。 </li><li>修剪含细胞沉淀凝固琼脂糖成一体的约1 - 用手术刀2毫米。含细胞沉淀琼脂糖片转移至新的1.5毫升管。 </li><li>通过添加和使用吸管不离心除去缓冲用600微升0.1M的二甲胂缓冲液的3倍 - 2洗净。使用含有用于下一步骤将细胞沉淀此琼脂糖片。在该步骤中，样品可以移动到下一个步骤之前过夜储存在4℃。 </li></ol> 2. Osmification，脱水，嵌入<ol><li>从1.5 ml管与含有琼脂糖用吸管将细胞沉淀除去二甲胂酸盐缓冲液中。加1.0毫升1％四氧化锇（OSO 4）溶液在通风橱中，并孵育在4℃下1小时。 </li><li>用600微升的0.1M二甲胂缓冲液洗三次，将样品与帽转移到20ml的玻璃闪烁瓶中。 </li><li>处理用于脱水，浸润样品和嵌入到树脂（ 例如 ，LX-112），在6孔板以下解决方案的系列变化。移动使用镊子小区沉淀琼脂糖片。 <ol><li>沉浸在RT在25％的乙醇的样品15分钟。 </li><li>沉浸在RT在50％乙醇的样品15分钟。 </li><li>浸入样品中75％的乙醇在室温15分钟。 </li><li>沉浸在RT在95％乙醇的样品15分钟。 </li><li>沉浸在RT在100％乙醇的样品15分钟，两次。 </li><li>浸入样品中乙醇:树脂（3:1）在RT下30分钟。 </li><li>浸入样品中乙醇:树脂（1:1）在RT下30分钟。 </li><li>浸入样品中乙醇:树脂（1:3）在RT 30分钟。 </li><li>沉浸在RT在纯树脂试样60分钟，两次。 </li></ol></li><li>将样品转移至小心使用镊子金字塔尖带状橡胶模具的底部，并添加在样品的顶部更纯树脂填充模具。孵育样品在60℃下72小时对树脂聚合。 </li><li>通过扭转橡胶模具中取出从模具中聚合的树脂金字塔。细胞的黑色小簇应在聚合树脂金字塔可见，如前面7所述。附加聚合的树脂金字塔使用氰基丙烯酸酯安装筒。 </li></ol><ol><li>修剪用刀片和一个超薄切片机安装块之后金刚石刀金字塔块。使与顶部和块平行的彼此底部的短宽的梯形形状，并与均匀地成角度侧面。金字塔的这种形状有助于对连续切片。 </li><li>此外，修剪与周围的钻石刀细胞沉淀块，并删除周围的细胞沉淀琼脂糖片塑料部分。 </li><li>使用超薄切片机切1微米的部分。移动从水中船1微米的部分使用睫毛工具，如先前报道7的金属环的工具载玻片。在载玻片上并转让滑动到热板（37℃）干燥转印部。 </li><li>添加的甲苯胺蓝溶液来使用装有0.22微米过滤器，并孵育3分钟的注射器的部分下降。用蒸馏水冲洗和L的幻灯片等章节干燥。检查用光学显微镜，以确定细胞的位置。 </li><li>切割超薄切片 - 使用钻石刀（70 100纳米）。移动2 - 从供水船200目网格3段使用睫毛工具的玻璃培养皿。 </li><li>传送包含与部分网格玻璃培养皿的热板上在30℃下干燥段为30分钟。 </li><li>染色以1％乙酸双氧铀滴网格在室温10分钟，然后在蒸馏水中漂洗6 - 8倍。染色与雷诺柠檬酸铅，在室温下5分钟，在蒸馏水中冲洗6 - 8倍。传送包含网格来的热板上的玻璃培养皿，在37℃干燥的部分。 </li><li>用电子显微镜分析的部分中，在80千伏7。 </li></ol>

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作者什么都没有透露。

该方法能够在低细胞数目TEM分析，通过使用伊文思蓝染色和琼脂糖嵌入脱水，树脂包埋期间本地化一个微小的细胞沉淀和切片工序。重要的是，它保持细胞簇一起，并保留在细胞的超结构，这是可取的，以检查数据的量化的多个小区。 在TEM研究，含有数百万个细胞的细胞团，常常需要用于有效埋入到琼脂糖/明胶基质和用于洗涤，脱水和渗透到从大量细胞中获得的数据。研究人员已经在一个BSA的双丙烯酰胺聚合树脂通过的细胞悬浮液的固定化，在薄支撑单层，或用在低细胞数目/稀少样品4-6 TEM分析细胞离心涂片的染色。然而，它仍然是一个冗长乏味的工作，以定位和切割的细胞在TEM下，因为细胞的基质中的稀疏分布的和细胞形态的可能改变，由于扩散和细胞离心涂片制备过程中，往往需要再嵌入切片前的小区之间的裂解。此外，单层仍然具有挑战性本地化的切片11。因此，即使从低细胞数量的稀少生物样品细胞沉淀为TEM分析1容易得多。 此方法表示在从有限的样本的定性和定量的超结构分析方面以前发表的方法4,5-一个显著优势。一起，该协议描述了一种有效的方式来定位使用伊文思蓝，这从低细胞数量容易使样本处理用于TEM启用定性和定量数据分析用于TEM细胞沉淀。通常情况下，用于TEM的样品制备需要，因为在样品处理F中的多个步骤的细胞比通常所需的光学显微镜的数量较多，或TEM和用于在合适的位置1,10切割单元块的正确方向。 已知的是定影后osmification提供图像的更好的对比度，特别是那些膜和糖原颗粒的电子显微镜9中 ，并且还支承染色醋酸铀，用于蛋白质的非特异性相对染色下，和柠檬酸铅，即污迹膜，核酸和糖原。然而，osmification几乎是不可能的，在悬浮稀少生物样品中的BSA-BA预嵌入后执行由于样品损失的切片和矩阵变暗期间的高可能性。因此，电子显微镜的质量在以往的研究4,9被攻破。这里Osmification导致与在琼脂糖的颜色变化最小细胞沉淀变黑。因此，本协议允许osmification后嵌入预琼脂糖，以提供在TEM IM良好的对比度年龄。 细胞簇很容易用这种方法电镜分析。预染色伊文思蓝帮助定位样品制备过程中微小的细胞团，而不影响子细胞器单元的详细信息。此方法提供了样品制备成功地从低细胞数进行TEM分析的另一种方法。 （ - 8微米5）在该方法中，使用小尺寸的电池被有效地进行TEM分析。因此，这种方法可以很容易地与较大的细胞，如成纤维细胞和内皮细胞的甚至更低的细胞数目（〜1000个细胞）通过。虽然Evans蓝不影响本研究的子细胞器的细胞细节，使用伊文思蓝与低细胞数免疫金电子显微镜的未经过测试。 一起，描述了样品制备从低细胞数进行TEM研究的有效方法。需要TEM研究揭示critica在形态和细胞的超结构l信息。因此，超结构信息经常缺少对各种稀有细胞群。这种选择和样品制备新方法将有助于从任何罕见的细胞群进行TEM研究。此外，这种方法允许从细胞团的分析定性和定量的数据。作为电子显微镜提供细胞超结构和组织的无与伦比的细节，此方法将是非常有用的，以了解对关键许多生物过程，但罕见的，细胞群。

细胞的超微结构和亚细胞器的细节已通过透射电子显微镜（TEM）从组织或细胞沉淀1,2-研究中主要显露。组织坚固件可以很容易地用于电子显微镜研究。然而，TEM分析1,3悬浮细胞仍然充满挑战和必要的高细胞数， 即数百万个细胞。正因为如此，在悬浮稀有细胞群的超结构分析， 例如，造血干细胞（HSC），不容易评估。对于使用细胞离心涂片或单层方法稀少样品TEM分析多次尝试未能从细胞的数量有限得到显著定量数据。因此，高泡孔数的要求限制了使用这一强大的工具的理解稀有细胞群的亚细胞超微结构的信息。 为TEM研究的一个重要的限制与细胞数目有限S IN悬挂是细胞处理的定位和由此，从有限的细胞，特别是小尺寸的TEM研究仍然充满挑战。几种替代方法已被采用，以对付这种限制:BSA /双丙烯酰胺（BSA-BA）细胞悬浮液的介导聚合，将细胞与染料以使它们在薄膜支持，包括盖玻片可见的染色， 透射电镜和从细胞离心制剂4-6分析。然而，非常有限的成功达到了，因为很少有细胞在超切片后各部分找到。识别稀疏细胞的主要挑战仍然存在，因为单层细胞保持在固化的凝胶切片大多不可见的。此外，细胞的细胞离心涂片器制剂可以改变它们的细胞组织由于细胞扩散和细胞结构的脆弱性。因此，这些固有的缺陷认股权证以罕见的细胞群与更多的执行TEM研究的新方法一致性。为了克服这个问题，我们已经描述了一种新的和替代的样品制备方法用于从稀有细胞群7 TEM研究。 这里，我们报告的有效样品制备协议从稀少生物样品具有一致的定性和定量的结果执行的TEM。固定后进行伊文思蓝染色来本地化来自低数量的细胞， 即 10000骨髓造血干细胞和祖细胞，否则仍然可见一个微小的细胞沉淀，并将沉淀物嵌入琼脂糖之前脱水和树脂包埋过程。此方法聚类细胞一起，使超微结构和造血干细胞的亚细胞组织（标识为林-的Sca-1 +的c-Kit +的Flt3 - CD34 - ; HSC）的有效分析，难得0.2 - 0.5％的细胞群在骨髓中。这一实验亲母育可以进行超结构的研究，并取得许多罕见但非常重要的群体的定量结果非常有用。

<table border="0" cellpadding="0" cellspacing="0" width="1096">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">Paraformaldehyde 20% Aqueous Soln</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">15713</td>
			<td style="width:440px;">CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">Gluteraldehyde 70% Aqueous Soln</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td>16350</td>
			<td>CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Cacodyladate buffer&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">12300</td>
			<td>Make 0.1 M Cacodyladate buffer (pH 7.4) in distilled water, store at 4&#176;C. CAUTION Irritant by inhalation or skin contact, wear gloves and work in fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evans Blue&#160;</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:113px;">E-2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low melting agarose&#160;</td>
			<td style="width:216px;">Sigma Aldrich</td>
			<td style="width:113px;">A-5030</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Osmium tetraoxide&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">19130</td>
			<td>1 gm in 50 ml of 0.1M Cacodyladate buffer to make 2% OsO4 stock, CAUTION Very toxic by inhalation or skin contact, strong oxidizer, wear protective clothing, eye protection, use fume hood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LX-112 Embedding Kits (LADD&#174;)</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">DDSA (Dodecenylsuccinic anhydride)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21340</td>
			<td>Mix 16% DDSA; 30% NMA; 54% LX-112 together and then add 1.4% DMP-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">NMA (Nadic methyl anhydride)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Epoxy Resins Epon 812 (LX-112)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21310</td>
			<td>CAUTION Toxic by inhalation, skin contact. Wear gloves, protective clothing, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">DMP-30 (2,4,6 -Tri(dimethylaminomethyl)phenol)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reynolds lead citrate (EM Stain II)</td>
			<td style="width:216px;">Leica</td>
			<td style="width:113px;"></td>
			<td>CAUTION Toxic by inhalation, avoid contact with skin or eyes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Uranyl acetate (EM Stain I)</td>
			<td style="width:216px;">Leica</td>
			<td style="width:113px;">8072820</td>
			<td>CAUTION Toxic by inhalation, avoid contact with skin or eyes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)&#160;</td>
			<td style="width:216px;">ATCC</td>
			<td style="width:113px;">30-2005</td>
			<td>composition: NaCl, NaHCO3, HEPES, Glucose, Ca, Mg, sod pyruvate etc. For for details https://www.atcc.org/~/media/Attachments/E/9/7/E/4893.ashx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyramid tip mold&#160;</td>
			<td>Ted Pella</td>
			<td style="width:113px;">10585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting cylinders</td>
			<td style="width:216px;">Ted Pella</td>
			<td style="width:113px;">10580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-microtome&#160;</td>
			<td style="width:216px;">(Leica EM UC7)</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">200 mesh grids (nikil)</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">G-200 Ni</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Electron Microscope</td>
			<td>Hitachi model H-7650&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Image capture engine software</td>
			<td>AMT-600&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm plastic petri dishes</td>
			<td>Fisher scientific&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Quick Bond Super glue Cyanoacrylate&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">72588</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintillation vials&#160;</td>
			<td>Fisher scientific&#160;</td>
			<td>03-337-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass slides&#160;</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eyelash tool</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal Loop tool&#160;</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

an alternative approach for sample preparation with low cell number for tem analysis

透射电子显微镜（TEM）提供了细胞组织和超微结构的细节。然而，稀有细胞群，尤其是细胞悬浮液，如造血干细胞（HSC）的TEM分析，仍然很有限，由于高细胞数的样品制备过程的要求。有用于从稀少样品TEM分析几个细胞离心涂片或单层的方法，但这些方法无法从细胞的数量有限得到显著定量数据。这里，对于在TEM研究的样品制备的替代的和新颖的方法是罕见的细胞群，使定量分析说明。 相对较低的细胞数量， 即10000造血干细胞，成功地应用于相比，数以百万计通常用于TEM研究细胞的TEM分析。特别是，多聚甲醛，戊二醛后进行伊文思蓝染色（PFA-GA）固定形象化狭小的牢房像素让，这有利于在琼脂糖嵌入。在超薄切片观察到大量细胞群。细胞有保存完好形貌，并高尔基复合体和几个线粒体的超结构细节也清晰可见。这种高效，容易和可重复的协议允许从低细胞数的样品制备和可用于对从有限的生物样品稀有细胞群的定性和定量TEM分析。

样品制备的高效和一致的协议来执行从描述的细胞数量低TEM分析。伊文思蓝和细胞转移到0.5 ml离心管后固定染色有助于可视化的微小细胞沉淀7。 Osmification在琼脂糖四氧化锇导致脱水和嵌入在一个简单的检测暗细胞沉淀。 从含有微小的细胞沉淀块半薄切片证实许多小区的集群一起（ 图1A和1B）。超薄切片从细胞聚集区切割并在电子显微镜下（ 图1C）进行分析。这些细胞的形态和超结构进行了很好地保存（ 图1D）。单个细胞的电子显微照片显示对细胞内高细节超结构（ 图1E）。此外，高倍率（50,000X）图像显示了保存完好的结构和亚细胞细胞器，诸如线粒体（ 图1F）的完整性。 <img alt="图1" src="/files/ftp_upload/54724/54724fig1.jpg" /> 图1: 图像在光镜和分析电子显微镜。 （A）的 1微米厚的光显微镜下甲苯胺蓝染色的部分的代表性领域的低放大率图像。（B）的光显微镜图像示出了簇细胞的1微米厚部分的甲苯胺蓝染色之后的完整的形态。 （℃）醋酸铀染色和柠檬酸铅后电子显微镜下观察到的小区集群的概述字段（D）的单个的LT-HSC电子显微镜下（E）高等MAGNIFHSC的ication图像显示的亚细胞结构。（F）从HSPC线粒体的高倍图像。比例尺:A / B，100微米; C，10微米; D，2微米; E / F，500纳米。 &#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54724/54724fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis at JoVE.com

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

an-alternative-approach-for-sample-preparation-with-low-cell-number

Nanyang Technological University

Research

JoVE Journal

Biology

13.6K 次观看.  Cincinnati Children’s Research Foundation. Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis. 

视频: An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

脱钙骨组织学，形态计量学和荧光染料分析制备

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

科研

生物学

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...