在本研究中，动物实验得到了加州大学旧金山分校机构动物护理和使用委员会的批准，并按照美国国立卫生研究院实验动物护理和使用指南进行。这里使用8-12周龄的成年C57BL / 6雄性和雌性小鼠（内部繁殖）。
1. DRG样品制备
<ol><li>用2.5%Avertin麻醉小鼠（参见材料表）。通过对疼痛刺激缺乏反应来确保充分麻醉。如前所述，用1x磷酸盐缓冲盐水（PBS）然后用4%甲醛经心灌注动物，如前所述14,15,16。</li>
	<li>从脊髓解剖 DRG 组织，如前所述14,15,16。</li>
	<li>在室温下将解剖的DRGs在4%甲醛中固定3小时。</li>
	<li>将DRGs置于30%蔗糖的PBS中，在4°C过夜。</li>
	<li>准备基础最佳切削温度 （OCT）<ol><li>加入OCT化合物（参见 材料表）以覆盖铝块的顶面，并在-20°C下冷冻5-10分钟（图1A）。</li>
		<li>使用低温恒温器（参见 材料表）切掉 OCT 的顶部，厚度为 30 μm，直到其表面平坦为基础 OCT（图 1B）。</li>
		<li>在将铝块从低温恒温器上取下之前，在底座 OCT 的底部（六点钟位置）做一个标记以标记其方向（图 1C）。</li>
		<li>在以下步骤中，将标记保持在六点钟位置以保持相同的方向，这可能会导致获得 DRG 样品的更多部分。 注意：如果失去方向并且以不同的角度切割 DRG 样品，则 DRG 样品无法从上到下完全切割，这将在基础 OCT 上留下一些样品并浪费，因为在尝试切割 DRG 样品时会遇到基础 OCT（图 1C）。</li>
	</ol></li>
	<li>执行 DRG 的 OCT 嵌入<ol><li>在将 DRG 组织放在 OCT 上之前将其擦干。<ol><li>在将 DRG 添加到模块上之前，确保去除样品周围的 30% 蔗糖/PBS 溶液。</li>
			<li>通过将DRG样品放在干燥的培养皿上干燥DRG样品（图2A），并用干镊子将其从一个位置移动到另一个位置两到三次（图2B）。</li>
			<li>在移动另一个DRG样品之前，用无绒组织擦干镊子（图2C）。</li>
		</ol></li>
		<li>将干燥的 DRG 放在底座 OCT 上。<ol><li>用干镊子将干燥的DRG放在12点钟位置的底座OCT上（图1D）。</li>
			<li>在 DRG 的上边缘和基础 OCT 的上边缘之间至少保持 5 毫米的距离。</li>
			<li>将DRG的背部放在12点钟位置，将腹侧放在6点钟位置（图1D）。</li>
		</ol></li>
		<li>用 OCT 包埋 DRG 组织。<ol><li>添加更多的OCT以圆形或椭圆形覆盖整个DRG样品，DRG位于中心（图1E）。</li>
			<li>确保 OCT 的上边缘覆盖基础 OCT 上边缘的 DRG（图 1F），因为这样可以更轻松地将截面转移到载玻片上（图 3A）。 注意：如果盖子 OCT 位于基础 OCT 的中间，则基础 OCT 的上边缘将阻挡载玻片以收集该部分（图 3B）。</li>
			<li>将覆盖的OCT和DRG样品在-20°C下再冷冻5分钟（图1G）。不要在 DRG 样品周围修剪 OCT。</li>
			<li>切掉 30 μm 厚的盖子 OCT 的顶部，直到 DRG 可见。</li>
		</ol></li>
	</ol></li>
</ol>2. DRG的冷冻切片
<ol><li>将低温恒温器截面厚度更改为12μm，将DRG截面切割到载玻片上（图1H）。</li>
	<li>用冷却至-20°C的小画笔将OCT部分保持在底部。 注意：重要的是不要完全切割整个部分，而是保留 1-3 毫米未切割（图 1I）。</li>
	<li>使用室温镊子的末端轻轻触摸该部分的底部（六点钟位置），使其粘在平台表面上（图 1J）。这样可以防止截面卷曲，从而难以在载玻片上收集截面。 注意：当截面的底部粘在平台表面上，并且截面的顶部仍然与盖子 OCT 连接时，该截面呈扁平形状（图 1K），这使得在载玻片上收集截面变得更加容易。</li>
	<li>取一个带电的载玻片（参见 材料表），然后慢慢地将载玻片放在切片上。一旦该部分开始粘在载玻片上，轻轻地向后拉载玻片（图1L）。</li>
	<li>按照相同的过程将更多的 DRG 部分放到载玻片上，而不会与这些部分重叠（图 1M）。确保新的 DRG 切片未放置在另一个 DRG 切片的 OCT 上（图 4），因为这样的切片不能很好地留在载玻片上，并且可以在免疫化学染色过程中被洗掉。</li>
</ol>3.载玻片上DRG部分的尼氏染色
<ol><li>在PBS和0.1%Triton X-10中洗涤切片10分钟。然后，用PBS洗涤切片两次。</li>
	<li>将 1：300 Nissl 染色剂（参见 材料表）涂在载玻片上，并在室温下孵育 20 分钟。</li>
	<li>用PBS加0.1%Triton X-100洗涤切片。然后，用PBS洗涤切片两次，每次5分钟，然后在室温下洗涤2小时。</li>
	<li>应用 4′，6-二脒基-2-苯基吲哚 （DAPI） fluoromount-G（参见 材料表）用盖玻片覆盖切片。</li>
</ol>

小鼠背根神经节冷冻切片

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作者没有什么可透露的。

该协议为小鼠DRG的低温恒温器切片提供了简单的分步程序，以可靠地在载玻片上获得高质量的DRG切片。该协议有四个关键步骤。首先，在将 DRG 样品放到基础 OCT 上之前，必须将 DRG 样品和镊子干燥。DRG 样品周围的任何液体都会在其周围形成冰壳，导致 DRG 部分与 OCT 分离并向上弯曲。其次，如果铝块没有标记，或者如果底座 OCT 覆盖了标记，则重要的是在底座 OCT 的底部（六点钟位置）做一个标记?...

背根神经节 （DRG） 包含初级感觉神经元、组织巨噬细胞和围绕初级感觉神经元的卫星细胞 1,2,3,4。它是处理无害和有害信号的关键解剖结构，在疼痛、瘙痒和各种周围神经疾病中起关键作用5、6、7、8、9、10、11、12、13。尽管已经开发了几种方法来解剖小鼠脊髓的 DRG 组织14,15,16，但由于 DRG 组织非常小，并且 DRG 样品的低温恒温器切片往往会弯曲成卷，因此很难将低温恒温器切片正确地转移到载玻片上。然而，DRG 组织的正确冷冻切片对于免疫组化研究和 DRG 感觉神经元的结构至关重要17、18、19、20、21、22、23。此外，由于单细胞RNA测序结果揭示了人类24和小鼠25中DRG感觉神经元的显着异质性，因此对DRG组织进行适当的冷冻切片对于研究不同DRG细胞在各种生理和病理条件下的功能作用至关重要。
尽管组织透明化技术已被应用于研究 DRG26 的 3D 重建，作为冷冻切除 DRG 的替代技术，但组织透明化技术既费时又费力。相比之下，DRG的冷冻切片快速且相对容易执行，因此它仍然是DRG和中枢神经系统其他区域的免疫组化和结构研究的关键技术。然而，在载玻片上获得高质量、完整和扁平的低温恒温器切片仍然是神经科学研究中的一个挑战，因为组织的样本量很小，如DRG和某些大脑区域，并且目前没有文章描述冷冻切片小尺寸组织样本的最佳方案，如小鼠DRG。
该协议为小鼠DRG的低温恒温器切片提供了一种简单的分步技术，以可靠地在载玻片上获得尽可能多的高质量DRG切片，用于后续的DRG研究。虽然该技术专为冷冻切除 DRG 样品而设计，但可用于冷冻切除样品量较小的各种其他组织。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Avertin</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-25G</td>
			<td>Anesthetize animal</td>
		</tr>
		<tr>
			<td>Epredia&#160;Cryotome Cryostat Cryocassettes, 25 mm dia. Crosshatched</td>
			<td>Fisherbrand</td>
			<td>1910</td>
			<td>Hold the OCT section at the bottom&#160;</td>
		</tr>
		<tr>
			<td>Ergo Tweezers</td>
			<td>Fisherbrand</td>
			<td>S95310</td>
			<td>Using the end of a tweezer to gently touch the bottom (6 o&#8217;clock) of the section so that it sticks to the platform surface to prevent the section from curving back in a roll&#160;</td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>1255015</td>
			<td>To collect the DRG section&#160;</td>
		</tr>
		<tr>
			<td>Marking pens</td>
			<td>Fisherbrand</td>
			<td>133794</td>
			<td>&#160;Mark the orientation of base OCT</td>
		</tr>
		<tr>
			<td>Scigen&#160;Tissue-Plus O.C.T. Compound</td>
			<td>Fisherbrand</td>
			<td>&#160;23730571</td>
			<td>Embedding medium for frozen tissue specimens to ensure optimal cutting temperature (O.C.T.).</td>
		</tr>
	</tbody>
</table>

a procedure for mouse dorsal root ganglion cryosectioning

在炎症和神经性疼痛、瘙痒以及其他周围神经系统疾病的研究中，高质量的小鼠背根神经节 （DRG） 低温恒温器切片对于适当的免疫化学染色和 RNAscope 研究至关重要。然而，由于 DRG 组织的样本量很小，因此在载玻片上始终如一地获得高质量、完整和扁平的低温恒温器切片仍然是一个挑战。到目前为止，还没有文章描述DRG冷冻切片的最佳方案。该协议提供了一种分步方法，以解决与DRG冷冻切片相关的常见困难。本文解释了如何从DRG组织样品中去除周围的液体，将DRG切片朝向相同的方向放置在载玻片上，并在不弯曲的情况下压平载玻片上的切片。尽管该协议是为冷冻切除DRG样品而开发的，但它可以应用于许多其他样品量小的组织冷冻切片。

The dorsal root ganglion (DRG) contains the primary sensory neurons, the tissue macrophages, and the satellite cells that surround the primary sensory neurons1,2,3,4. It is a key anatomic structure in processing innocuous and noxious signals, and plays critical roles in pain, itch, and various peripheral nerve disorders5,6,7,8,9,10,11,12,13. Although several methods have been developed to dissect DRG tissue from the mouse spinal cord14,15,16, cryosectioning of the DRG tissue remains challenging as the DRG tissue is quite small, and cryostat sections of DRG samples tend to curve into rolls, making it difficult to properly transfer the cryostat sections onto glass slides. However, proper cryosectioning of the DRG tissue is crucial for immunohistochemistry studies and the structure of DRG sensory neurons17,18,19,20,21,22,23. Moreover, as single-cell RNA sequencing results have revealed the remarkable heterogeneity of DRG sensory neurons in both humans24 and mice25, proper cryosectioning of DRG tissue is critical for investigating the functional role of different DRG cells in various physiological and pathological conditions.
Although the tissue-clearing technique has been applied to investigate the 3D reconstruction of the DRG26 as an alternative technique of cryosectioning the DRG, the tissue-clearing technique is time- and labor-consuming. In comparison, cryosectioning of the DRG is quick and relatively easy to perform, and thus it remains to be a key technique for immunohistochemistry and structure studies of the DRG and other regions of the central nervous system. However, obtaining high-quality, intact, and flat cryostat sections onto glass slides remains to be a challenge in neuroscience research because of the tiny sample size of tissues, like the DRG and certain brain regions, and there is no article describing the optimal protocol at this point for cryosectioning small-sized tissue samples, such as mouse DRGs.
This protocol provides an easy, step-by-step technique for cryostat sectioning of the mouse DRG to reliably obtain as many high-quality DRG sections on the slides for subsequent DRG studies. While specifically designed for cryosectioning DRG samples, this technique can potentially be used for cryosectioning various other tissues with a small sample size.

作者聚焦：小鼠背根神经节冷冻切片程序  

小鼠背根神经节冷冻切片的程序

It remains a challenge to consistently obtain high-quality, intact, and flat mouse dorsal root ganglion cryostat sections. The research aims to answer how to acquire ideal mouse dorsal root ganglion cryostat section. This protocol provides an easy step-by-step technique for cryostat section of the mouse DRG to high quality, intact, and flat mouse DRG cryostat sections.This presented article explains how to remove surrounding liquid from the mouse DRG sample tissue, placing the DRG sections on the slides facing the same orientation, and flatten the DRG sections without curving up. One major challenge is that the OCT sections of mouse DRG tend to curve into a roll, making it difficult to place onto the glass slide. It is also challenging to acquire sufficient sections from one sample due to the small size of the mouse DRG.

目前的研究从一只小鼠 L4 DRG 中收集了大约 16 个连续的高质量 DRG 切片。获得的部分没有任何失真。 图 1 描述了冷冻切片的分步过程。从组织切片中去除多余的液体如图 2 所示。 OCT包埋组织的过程如图3所示。 图 4 显示了载玻片上 DRG 部分的正确位置。 图5 显示了小鼠DRG切...

Watch this Scientific Journal Video about A Procedure for Mouse Dorsal Root Ganglion Cryosectioning at JoVE.com

这里介绍的是持续获得高质量背根神经节低温恒温器切片的发展。

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...

始终如一地获得高质量、完整和扁平的小鼠背根神经节低温恒温器切片仍然是一个挑战。该研究旨在回答如何获得理想的小鼠背根神经节低温恒温器切片。该协议提供了一种简单的分步技术，用于小鼠DRG的低温恒温器切片到高质量，完整和扁平的小鼠DRG低温恒温器切片。本文解释了如何从小鼠DRG样品组织中去除周围的液体，将DRG切片放置在面向相同方向的载玻片上，并在不弯曲的情况下压平DRG切片。一个主要的挑战是鼠标 DRG 的 OCT 部分往往会弯曲成卷状，因此很难放置在载玻片上。由于小鼠DRG的尺寸较小，从一个样品中获取足够的切片也具有挑战性。

a-procedure-for-mouse-dorsal-root-ganglion-cryosectioning

Research

JoVE Journal

Neuroscience

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

3.9K 次观看.  Capital Medical University. 始终如一地获得高质量、完整和扁平的小鼠背根神经节低温恒温器切片仍然是一个挑战。该研究旨在回答如何获得理想的小鼠背根神经节低温恒温器切片。该协议提供了一种简单的分步技术，用于小鼠DRG的低温恒温器切片到高质量，完整和扁平的小鼠DRG低温恒温器切片。本文解释了如何从小鼠DRG样品组织中去除周围的液体，将DRG切片放置在面向相同方向的载玻片上，并?...