这项工作得到了中国国家自然科学基金 (31400936、31460260号赠款) 和江西省自然科学基金 (20171BAB215020) 的支持。我们还感谢南昌大学与伦敦玛丽大学的联合项目支持这项工作。

这里介绍的所有方法均经南昌大学动物保育委员会批准。
1. 装配设备并连接切片
<ol><li>根据要求设计参数 (补充图 1)。</li>
	<li>将参数提交当地工厂生产丙烯酸板。</li>
	<li>按顺序装配所有部件: 使用氯仿将7个商业丙烯酸板组合成一个带有截面通道和水浴的水箱 (图 1)。 注意: 氯仿在遇到光和氧时会产生有毒物质。把器具装配在通风罩里。</li>
	<li>将管状电热元件通过压克力板 #1 (图 2A、箭头) 安装在孔中。</li>
	<li>要安装温度控制器, 请使用电钻在 #2 丙烯酸板上创建一个小孔, 并安装温度检测器 (图 2A、箭头)。在水箱上安装数字恒温器 (图 2A)。 注: 温度探测器应在水面以下1厘米以内。</li>
	<li>要提供电源接入, 请在打开设备前调整电压低于 24 V。</li>
	<li>从切片中移除剖面废物托盘。</li>
	<li>用中性硅胶密封胶将截面通道与切片刀片架连接 (图 2B)。 注: 请勿涂抹在刀片顶部上方的中性硅胶密封胶, 使旧刀片易于更换。</li>
	<li>夜间干燥, 允许氯仿和中性硅酮密封胶时间固化。</li>
	<li>将刀片式服务器安装到刀片式服务器上。将水倒入水浴中 (图 2C)。水面应该只是触摸刀片的顶部 (图 2D,箭头头)。</li>
</ol>2. 石蜡切片
注: 碎屑经常堆积在蜡块和水面的上缘或下边缘。这些碎片应定期清除。
<ol><li>
执行常规石蜡切片10。
	<ol>
<li>将石蜡嵌入试样夹在旋转切片上, 将刀片置于刀片夹持器中。</li>
		<li>转动手轮和切片试样。</li>
		<li>将石蜡切片的丝带转移到38.5 摄氏度温水浴。</li>
		<li>展开后, 从水面上的显微镜上把切片捡起来。</li>
		<li>将烤箱中的幻灯片干燥42摄氏度。</li>
	</ol>
</li>
	<li>
改进石蜡切片 (图 3)
	<ol>
<li>打开设备。</li>
		<li>打开加热器与温度检测开关, 并设置目标温度之间的38.0 °c 到40.0 °c, 提前1小时, 以温暖的水。</li>
		<li>修改石蜡嵌段获得的石蜡块;然后把石蜡块放在切片的卡带钳上, 然后锁上。 注: 应水平调整石蜡块的上、下边界, 防止截面带状形成弧形, 无法流经狭窄断面通道。</li>
		<li>通过剪切较厚的部分快速接近样品。</li>
		<li>调整到合适的断面厚度 (10 µm 成年小鼠大脑, 成年小鼠肾脏, 胚胎小鼠大脑, 4 µm 为斑马鱼的眼睛)。转动手轮, 各部分将自动进入截面通道和浴缸。</li>
		<li>切片的标本在一个缓慢和均匀的切割冲程 (图 3A)。 注: 第一节的 Thethickness 将在手轮停止几分钟后改变。当切片有连续划痕时, 应更换一次性刀片。</li>
		<li>将各部分引导至浴腔内以直线浮动 (图 3B、3C)。 注: 如果头部部分附着在腔壁上, 请使用画笔导轨使其浮动。</li>
		<li>将部分粘贴到显微镜幻灯片上。在水浴中充分展开部分后, 用镊子分隔几个部分。然后将它们从水浴中接上显微镜幻灯片 (图 3D)。 注: 应从水浴后部拾起部分, 因为部分通道和水浴的前部仍未完全展开。</li>
		<li>把这些幻灯片放在一个42摄氏度的烤箱里过夜。然后可以无限期地将幻灯片存储在室温下。</li>
	</ol>
</li>
	<li>应用苏木精-伊红 (he) 染色, 以评价改进方法8。</li>
</ol>

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<li>Sabaliauskas, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+zebrafish+histology%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">High-throughput zebrafish histology.</a> Methods. 39 (3), 246-254 (2006).</li>
<li>Chen, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hybrid-Cut%3A+An+Improved+Sectioning+Method+for+Recalcitrant+Plant+Tissue+Samples%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples.</a> Journal of Visualized Experiments. (117), e54754(2016).</li>
<li>Kucherenko, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paraffin-Embedded+and+Frozen+Sections+of+Drosophila+Adult+Muscles%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles.</a> Journal of Visualized Experiments. (46), e2438(2010).</li>
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<li>Tucker, D. K., Foley, J. F., Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sectioning+Mammary+Gland+Whole+Mounts+for+Lesion+Identification%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Sectioning Mammary Gland Whole Mounts for Lesion Identification.</a> Journal of Visualized Experiments. (125), e55796(2017).</li>
<li>Venegas-Pino, D. E., Banko, N., Khan, M. I., Shi, Y., Werstuck, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+Analysis+and+Characterization+of+Atherosclerotic+Lesions+in+the+Murine+Aortic+Sinus%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus.</a> Journal of Visualized Experiments. (82), e50933(2013).</li>
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作者在这一设备上有专利, 并且声明没有竞争利益。

为改善石蜡切片形貌, 解决常规石蜡切片过程中的浪费时间问题, 我们建立了一种改进的石蜡切片方法, 结合切割和展开。这种改进的方法依赖于简单的设备, 包括剖面通道, 水浴和加热器与温度检测开关。节带通过剖面通道进入水浴, 并在切割时自动展开。因此, 该方法提高了石蜡切片的质量和效率, 这是通过石蜡切片的成年小鼠海马, 成年小鼠肾脏, E15.5 小鼠的大脑, 和成年斑马鱼眼睛验证。
为了避免在常规石蜡切片期间卷曲, 许多调整, 如滋润组织表面, 冷却蜡块, 增加湿度, 并结合 cryosectioning6,7, 8;然而, 这些调整使石蜡切片更繁琐。一个依赖于流体水的商业部门转移系统更加成功。在该系统中, 该剖面在切割后立即接触到水面, 电机驱动的水流将截面从刀片持有者转移到下温水浴9。由于其机理, 静电不会抑制切片, 从而提高断面质量。此外, 由于该系统避免手动将部分从刀片持有人转移到水浴, 容易损坏易碎的部分, 它也有助于获得高品质的更薄的部分。
通过改进的方法, 简单的设备借鉴了商业断面传输系统的经验, 具有一定的优越性。首先, 设备比较便宜, 可以在开发区域的实验室推广这种方法, 可以补充商业部门的转移系统。第二, 设备中的水是静止的, 不会干扰部分, 从而可以获得较薄的部分。采用改进的方法, 对斑马鱼的2µm 厚度进行了连续切片, 获得的切片质量较高。第三, 在设备中使用较大的水浴水箱, 允许至少40节同时展开和浮动, 允许两个操作者进行合作。一人可以连续切下组织标本, 另一种可以直接将切片钓上来。这样可以减少等待节展开的时间。连续切割与均匀切削冲程也改善剖面质量。
我们的简单设备的结构和外观需要改进。例如, 组件 (如电源、加热器和温度控制器) 可以集成, 以使此设备更加完整和便携。此外, 商用旋转 microtomes 有不同的大小。要连接到其他 microtomes, 应调整该设备的角度和高度。石蜡切片结合其他分子技术广泛应用于神经生物学、组织形态学等研究领域11、12、13。这种方法有可能提高临床检查效率14,15和其他生物标本超过三种组织类型在这里使用。

形态学研究在生物研究中具有重要意义。虽然新技术允许研究人员直接从整个组织或有机体观察他们的目标1,2,3, 切割标本成薄切片, 其次是染色, 仍然是主要的方法 fornot 组织形态学, 也可直接在组织中进行蛋白靶向。光镜使用三切片类型: 石蜡, 冷冻和半薄切片。虽然 cryosectioning 是常见的保护组织抗原性, 标本准备是简单的, 保留组织形态学较差, 不适合薄切片4,5。石蜡切片是展示保存完好的形态学最常用的方法。由于试样完全脱水, 并嵌入蜡, 石蜡块可以无限期储存。此外, 石蜡切片产生薄切片, 以改善生物探针进入进一步的实验和减少细胞层叠加在 Z 方向。
然而, 传统的石蜡切片是繁琐的, 需要操作员的技能。石蜡切片经过固定, 脱水, 嵌入, 切割和漂浮。重要的是, 将节带从刀座转移到水浴是必要的, 但对于初级操作者来说是困难的。特别是在干燥的空气中, 节带会由于静电而扭曲, 在温暖的水面上很难展开。为提高切片质量, 在切片刀片通过后, 将所暴露的组织表面湿润, 将蜡块浸泡在冰水中, 或者在切片附近用加湿器提高湿度, 建议6、7.改进石蜡切片的新方法包括混合石蜡嵌入, cryosectioning8和商业部分转移系统协助9。虽然这些方法部分提高了石蜡切片的速度和质量, 但它们使切片变得更加繁琐, 商业剖面转移系统是昂贵的。
在本协议中, 我们演示如何逐步创建简单, 廉价和灵活的设备, 这可以连接到旋转切片的刀片持有人。该设备由截面通道、水浴和带有温度检测开关的加热器组成。切割后, 大量的断面流进断面通道, 直接进入水浴, 从而自动展开。这提高了石蜡切片的效率, 使该技术更方便。使用这种方法, 更多的成年小鼠海马切片, 成年小鼠肾脏切片, 胚胎15.5 天老 (E15.5) 小鼠脑切片, 和成年斑马鱼眼切片被收获在较少的时间, 并保持更完整的形态。这种方法也可以用于其他组织样品, 需要加速石蜡切片, 同时避免损失的部分区分。

<table><tbody><tr><td>Incubator</td><td>Boekel Scientific</td><td>133000-2</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>64-17-5</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>1330-20-7</td><td></td></tr><tr><td>Paraplast</td><td>Leica</td><td>39601006</td><td></td></tr><tr><td>Heated Paraffin Embedding Module</td><td>Leica</td><td></td><td></td></tr><tr><td>Commercial acrylic board</td><td></td><td></td><td></td></tr><tr><td>Trichloromethane</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>67-66-3</td><td></td></tr><tr><td>Tubular electric heating element(12 V 200 W)</td><td></td><td></td><td></td></tr><tr><td>Temperature controller(12 V 120 W)</td><td>Mingsuo</td><td>XH-W3002</td><td></td></tr><tr><td>Rotary microtome&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Neutral silicone sealant</td><td></td><td></td><td>Link the water channel with the microtome knife holder</td></tr><tr><td>Voltage transformer</td><td>Dearll</td><td>S-250-12</td><td></td></tr><tr><td>Disposable blade</td><td>Accu-Edge</td><td>4689</td><td></td></tr><tr><td>Hematoxylin</td><td>Baso Diagnostics Inc.</td><td>BA-4025</td><td></td></tr><tr><td>Eosin&amp;nbsp;</td><td>Baso Diagnostics Inc.</td><td>BA-4025</td><td></td></tr><tr><td>Microslide</td><td>Sail Brand</td><td>7105</td><td></td></tr><tr><td>Neutral balsam</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>10004160</td><td></td></tr><tr><td>Coverslip&amp;nbsp;</td><td>Citoglas</td><td>10212424C</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td>Xilong Chemical</td><td>7647-01-0</td><td></td></tr><tr><td>Water bath for paraffin sections</td><td>Leica</td><td></td><td></td></tr><tr><td>HistoCore Arcadia C&amp;nbsp;- Cold Plate</td><td>Leica</td><td></td><td></td></tr><tr><td>paraffin repellent spray&amp;nbsp;</td><td>Thermo Scientific</td><td>9990420</td><td></td></tr></tbody></table>

the cutting and floating method for paraffin-embedded tissue for sectioning

石蜡嵌入组织切片被广泛应用于组织学和病理学。然而, 这是乏味的。为了改进这种方法, 几家商业公司利用流体水设计了复杂的断面传输系统。为了简化这项技术, 我们创建了一个简单的方法, 使用自制的设备, 结合切割和浮动在一个简单的恒温室;因此, 各部分自动进入水浴在水面上。用常规石蜡切片和所提出的方法进行比较, 对成年小鼠脑、成年小鼠肾脏、胚胎小鼠大脑和成年斑马鱼眼的海马体进行切割。统计分析表明, 改进后的方法节省了时间, 产生了较高的质量剖面。此外, 在短时间内石蜡切片的整个标本是容易的初级操作者。

改进后的方法增加了完整石蜡切片的数量。我们对成年小鼠海马组织、成年小鼠肾脏、胚胎小鼠大脑和斑马鱼的眼睛进行了新的检测。水被增加到坦克, 并且水温保持在38.0 °c 之间到40.0 °c。在连续制备组织样品后, 切片并与常规切片进行比较。该方法避免了切片丢失, 增加了成年小鼠海马、成年小鼠肾脏、E15.5 鼠脑和成年斑马鱼眼中完整切片的比例 (图 4A、4C、4E、4G)。最后, 我们的方法改进了切片, 防止切片损失和收获更完整的切片每标本。
改进后的方法减少了试样的花费时间。在比较常规方法和改进方法之间的时间时, 我们对成年小鼠海马、成年小鼠肾脏、E15.5 鼠脑和成人进行了全系列切片 (从第一节到器官的最后一节)。斑马鱼的眼睛。结果表明, 改进后的方法在所有测试样品中加速了石蜡切片速度 (图 4B、4D、4F、4H)。一般而言, 我们的方法允许更快的高质量石蜡切片。
用 H 和 E 染色观察了斑马鱼视网膜的一系列切片。对于斑马鱼视网膜的视神经盘段的序列切片, 从改良方法中提取的切片比常规石蜡切片法更好的完整性 (图 5A、5B)。这两种方法对大多数收获部分的质量没有显著差异 (图 5C、5D)。有时, 从传统方法中收获的部分的质量相对较低 (图 5E、5F)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58288/58288fig1.jpg"> 图 1: 将7个商业丙烯酸板组装成罐体的过程.(一) 7 个亚克力板的整体看法。(B G)从1号板到板 #7 的水箱逐步组装。(H) 装配后的主水箱。(一) 一般水箱尺寸;卸下罐面上的保护膜进行进一步安装。l = 400 毫米, l ' = 220 毫米, H = 122 毫米, W = 200 毫米.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58288/58288fig1large.jpg" target="_blank">请单击此处查看此图的较大版本</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58288/58288fig2.jpg"> 图 2: 适当组装好的设备, 供使用.(A) 连接到旋转切片后的完好设备。箭头指示, 温度探测器通过电钻安装在小孔上。箭头头, 加热器 (管式电热元件) 安装在板孔 #1。(B) 放大的虚线区域, 切片刀片持有者与剖面通道之间的连接特征。箭头头, 刀片持有人和部分渠道连接的中性硅胶密封剂。(C) 与 B 相同, 浇水和刀片安装。(D) 在 c 箭头头的虚线区域的侧面视图, 水面接近刀片边缘。: 电压互感器;世界银行: 水浴;DT: 数字恒温器;B: 刀片;BH: 刀片持有人;SC: 节频道。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58288/58288fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58288/58288fig3.jpg"> 图 3: 改进石蜡切片工艺.位于左下角的数字分别为放大的虚线区域。箭头指示, 第一个部分。(a、a)截割后立即直接进入剖面通道。(b、b)节带穿过剖面通道和水浴之间的边界。(c、c)剖面漂浮到水浴的后部, 并充分展开。(d、d)当它们到达水浴的后部并充分展开时, 这些切片会被显微镜下的幻灯片所拾取。SC: 区段渠道;世界银行, 水浴。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58288/58288fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58288/58288fig4.jpg"> 图 4: 改进的切片方法提高了全系列断面的效率和成品率.屈服率相当于所有切口部分完整截面的比例。数据显示为均值电子扫描电镜并由同一操作者获取。(A、C、E、G)改进的方法防止了成年小鼠海马段的丢失 (91.04 1.429% N = 3, vs 97.63, 0.4864% N = 3, p = 0.0120), 成年小鼠肾脏 (92.30 @ 0.5689% n = 3, vs 95.99) 0.7055% n = 3, p = 0.0153), E15.5 鼠脑 (90.67 @ 1.549%, N = 3 vs 97.01 0.2816%, N = 3, p = 0.0158), 和成年斑马鱼眼睛 (91.27 @ 0.9734% n = 4 vs 96.32, 0.7217% n = 4, p = 0.0059)。(B、D、F、H)改进的方法节省时间与常规方法相比, 成年小鼠海马 (126.0 @ 3.606 分钟vs 79.67 @ 1.856 分钟, N = 3, p = 0.0003), 成年小鼠肾脏 (66.67 @ 1.764 分钟vs 41.33 * 2.186 分钟, N = 3, p = 0.0008), E15.5 鼠标 br第 (99.67) 3.480 分钟vs 52.33 @ 4.333 分钟, N = 3, p = 0.0010), 和成年斑马鱼眼睛 (48.00 @ 2.160 分钟vs 30.75, 1.797 分钟, N = 4, p = 0.0009)。* p < 0.05, ** p < 0.01, *** p < 0.005。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58288/58288fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58288/58288fig5.jpg"> 图 5: 对从常规方法获得的斑马鱼眼中的全系列部分的 H 和 E 染色进行比较, 并对同一操作者的石蜡切片进行改进.(A) 常规石蜡切片 (21 节)。(B) 改进石蜡切片 (24 节)。(C) A6 的虚线区域。(D) B7 的虚线区域。(E) A17 的虚线区域。(F) B19 的虚线区域。星号, 部分质量相对较差。箭头头, 视盘;箭, 视网膜组织的严重撕裂。刻度条 = 200 µm (A 和 B);刻度条 = 50 µm (C、D、E、F)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58288/58288fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图 1: 7 商业丙烯酸板的尺寸参数.该数字表示相应板边距的长度。单位: mm,<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58288/supplementary_figure.AI">请点击这里下载此文件.</a>

Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

切片中石蜡嵌入组织的切割和漂浮方法

在这里, 我们提出了一个改进石蜡切片的协议。这种方法结合切割和浮动使用一个简单的恒温室, 以避免转让过程中所需的传统方法。结果表明, 石蜡切片的效率和数量均有显著提高。

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

the-cutting-floating-method-for-paraffin-embedded-tissue-for

Nanyang Technological University

Research

JoVE Journal

Neuroscience

18.3K 次观看.  Nanchang University. 在这里, 我们提出了一个改进石蜡切片的协议。这种方法结合切割和浮动使用一个简单的恒温室, 以避免转让过程中所需的传统方法。结果表明, 石蜡切片的效率和数量均有显著提高。

视频: The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

薄切片进行免疫组化切片的筹备工作

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

科研

生物学

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is presented.

非交联无甲醛组织固定剂 HER2 鱼的协议, 结合低温保存和福尔马林固色的优点

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its ...

癌症研究

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

微生物菌落生物膜的石蜡嵌入和薄切片显微分析

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

免疫与感染

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

医学

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

肿瘤切片提取物的建立与分析是诊断检测的前提

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割:用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

胰岛嵌段石蜡切片

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

<meta charset="utf8"></head><body>优化的薄子宫内膜小鼠模型，死亡率低，粘连最小

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

<meta charset="utf8"></head><body>建立薄子宫内膜的小鼠模型

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

建立薄子宫内膜的小鼠模型

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

切片中石蜡嵌入组织的切割和漂浮方法

摘要

探索更多视频

薄切片进行免疫组化切片的筹备工作

非交联无甲醛组织固定剂 HER2 鱼的协议, 结合低温保存和福尔马林固色的优点

微生物菌落生物膜的石蜡嵌入和薄切片显微分析

移植到小鼠卵巢脂肪垫

肿瘤切片提取物的建立与分析是诊断检测的前提

混合式切割:用于顽拗植物组织样品的改进切法

胰岛嵌段石蜡切片

建立薄子宫内膜的小鼠模型

建立薄子宫内膜的小鼠模型

建立薄子宫内膜的小鼠模型