Name: combining multiplex fluorescence in situ hybridization with fluorescent immunohistochemistry on fresh frozen or fixed mouse brain sections
Uploaded: 2021-06-25T13:00:00
Description: Watch this Scientific Journal Video about Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections at JoVE.com

这项工作由澳大利亚研究委员会发现项目资助DP180101890和Rebecca L Cooper医学研究基金会项目资助PG2018110

组织预处理步骤的摘要见 图1。所有程序均按照新南威尔士大学动物护理和伦理委员会根据科学目的使用和护理动物的指南（澳大利亚国家健康与医学研究委员会）进行。
1.新鲜冷冻脑组织的样品制备
<ol><li>经心灌注<ol><li>制备肝素化 （2500 U/L） 0.1 M 磷酸盐缓冲液 （PB），pH 7.5。通过将干冰与乙醇混合制成干冰乙醇浆。这将具有大约-72°C的温度，并将?...

<ol>
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在神经科学中，FISH 和 IHC 通常用于研究神经元亚群中 mRNA 或蛋白质的空间组织和功能意义。本研究中描述的方案增强了同时检测脑切片中mRNA和蛋白质的能力。我们的多重 FISH-IHC 联合检测能够在新鲜冷冻和固定脑制剂中对 NTS 中不同的神经元亚群进行表型鉴定。固定冷冻组织制剂中的 FISH-IHC 产生了可靠的 IHC 结果。例如，低丰度和高丰度 mRNA（分别为 GalR1 和 GlyT2）和 IHC（靶向酪氨酸羟化酶）的多?...

神经化学定义神经元亚群的蛋白质和 mRNA 通常分别通过免疫组织化学 （IHC） 和/或 原位 杂交 （ISH） 的组合进行鉴定。将 ISH 与 IHC 技术相结合，通过最大限度地提高多重标记能力，促进了功能神经元特有的共定位模式（神经化学编码）的表征。
与早期的RNA检测方法（如放射性ISH和非放射性显色ISH）相比，包括RNAscope在内的荧光ISH（FISH）方法具有更高的灵敏度和特异性。FISH 可将单个 mRNA 转录本可视化为点状染色斑点1。此外，RNAscope检测允许使用不同的荧光团标签一次标记更多数量的RNA靶标。尽管有这些优点，但技术限制可能会影响可用于单个实验的荧光基团/显色剂的数量。其中包括显微镜滤光片组的可用性;与单独使用每种技术相比，当神经化学鉴定联合使用 FISH 和 IHC 时，这些考虑会更加复杂，因为一种方法的最佳固有步骤可能对另一种方法有害。
先前FISH联合IHC的应用已经证明了在人B细胞淋巴瘤2、鸡胚胎3、斑马鱼胚胎4、小鼠视网膜5和小鼠内耳细胞6中特异性细胞靶点的表达。在这些研究中，组织制备是福尔马林固定石蜡包埋 （FFPE）2,3,5 或新鲜的全镶嵌 4,6。其他研究将显色RNAscope应用于固定的小鼠和大鼠脑制剂7,8,9。特别是，Baleriola 等人。8 描述了用于联合 ISH-IHC 的两种不同组织制剂;固定小鼠脑切片和FFPE人脑切片。在最近的一篇出版物中，我们将 FISH 和荧光 IHC 结合在新鲜冷冻切片上，同时可视化脑干网状结构中的低丰度 mRNA（甘丙肽受体 1，GalR1）、高丰度 mRNA（甘氨酸转运蛋白 2，GlyT2）和囊泡乙酰胆碱转运蛋白 （vAChT） 蛋白10。
孤束核 （NTS） 是参与自主神经功能的主要大脑区域。这种异质的神经元群位于后脑，接收并整合了大量的自主神经信号，包括调节呼吸的信号。NTS 包含多个神经元群，其表型特征可能是 mRNA 靶标的表达模式，包括 GalR1 和 GlyT2，以及酪氨酸羟化酶 （TH） 和转录因子配对样同源盒 2b （Phox2b） 的蛋白质标记物。
RNAscope所有者推荐新鲜的冷冻组织制剂，但通过全动物经心灌注固定制备的组织，以及固定冷冻组织切片的长期冷冻保护（在-20°C下储存）在许多实验室中很常见。因此，我们试图使用新鲜冷冻和固定冷冻组织制剂建立FISH与IHC联合使用的方案。在这里，我们提供了新鲜冷冻和固定冷冻的脑切片：（1）FISH和荧光IHC联合的方案（2）使用每种制剂时产生的mRNA和蛋白质标记的质量的描述（3）描述NTS中GalR1和GlyT2的表达。
我们的研究表明，当与RNAscope方法结合使用时，IHC的成功率在新鲜冷冻和固定冷冻制剂中各不相同，并且取决于细胞内靶蛋白的定位。在我们手中，膜结合蛋白标记总是成功的。相比之下，即使在细胞质蛋白在转基因动物中过表达的情况下，细胞质蛋白的 IHC 也需要进行故障排除 （Phox2b-GFP）11。最后，虽然 GalR1 在 NTS 的非儿茶酚胺能神经元中表达，但 GlyT2 在 NTS 中不存在表达。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ANIMALS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mouse</td>
			<td>Australian BioResources, Moss Vale</td>
			<td>MGI: 2159769</td>
			<td></td>
		</tr>
		<tr>
			<td>Phox2b-eGFP mouse</td>
			<td>Australian BioResources, Moss Vale</td>
			<td>MGI: 5776545</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate</td>
			<td>Loctite</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Sigma-Aldrich</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-Sodium</td>
			<td>Clifford Hallam Healthcare</td>
			<td>1070760</td>
			<td>Consult local veterinary supplier or pharmacy.</td>
		</tr>
		<tr>
			<td>Lethabarb (Sodium Pentabarbitol) Euthanasia Injection</td>
			<td>Virbac (Australia) Pty Ltd</td>
			<td>N/A</td>
			<td>Consult a veterinarian for local pharmaceutical regulations regarding Sodium Pentabarbitol</td>
		</tr>
		<tr>
			<td>Molecular grade agarose powder</td>
			<td>Sigma Aldrich</td>
			<td>5077</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound, 118mL</td>
			<td>Scigen Ltd</td>
			<td>4586</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, prilled, 95%</td>
			<td>Sigma-Aldrich</td>
			<td>441244-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone, average mol wt 40,000&#160; (PVP-40)</td>
			<td>Sigma-Aldrich</td>
			<td>PVP40</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>With or without DAPI</td>
		</tr>
		<tr>
			<td>RNAscope Multiplex Fluorescent Reagent Kit (up to 3-plex capability)</td>
			<td>Advanced Cell Diagnostics, Inc. (ACD Bio)</td>
			<td>ADV320850</td>
			<td>Includes 50x Wash buffer and Protease III</td>
		</tr>
		<tr>
			<td>RNase Away</td>
			<td>Thermo-Fisher Scientific</td>
			<td>7003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Sigma-Aldrich</td>
			<td>252859</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20, for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop incubator</td>
			<td>Thermoline scientific micro incubator</td>
			<td>Model: TEI-13G</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Matrix, Mouse, 30g Adult, Coronal, 1mm</td>
			<td>Ted Pella</td>
			<td>15050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Drawing-up needle (23 inch gauge)</td>
			<td>BD</td>
			<td>0288U07</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic Barrier Pen</td>
			<td>Vector labs</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech Science Kimwipes Delicate Task Wipes</td>
			<td>Kimberley Clark Professional</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX51</td>
			<td>Olympus</td>
			<td>BX-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Coleparmer Masterflex</td>
			<td>L/S Series&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Retiga 2000R Digital Camera</td>
			<td>QImaging</td>
			<td>RET-2000R-F-CLR</td>
			<td>colour camera</td>
		</tr>
		<tr>
			<td>SuperFrost Plus Glass Slides (White)</td>
			<td>Thermo-Fisher Scientific</td>
			<td>4951PLUS4</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating Microtome (Vibratome)</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman qualitative filter paper, Grade 1, 110 mm diameter</td>
			<td>Merck</td>
			<td>WHA1001110</td>
			<td></td>
		</tr>
		<tr>
			<td>SOFTWARES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CorelDRAW&#160;</td>
			<td>Corel Corporation</td>
			<td>Version 7</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI (ImageJ Distribution)</td>
			<td>Open Source/GNU General Public Licence (GPL)</td>
			<td>N/A</td>
			<td>ImageJ 2.x: Rueden, C. T.; Schindelin, J. &#38; Hiner, M. C. et al. (2017), &#34;ImageJ2: ImageJ for the next generation of scientific image data&#34;, BMC Bioinformatics 18:529, PMID 29187165, doi:10.1186/s12859-017-1934-z&#160;&#160; and Fiji: Schindelin, J.; Arganda-Carreras, I. &#38; Frise, E. et al. (2012), &#34;Fiji: an open-source platform for biological-image analysis&#34;, Nature methods 9(7): 676-682, PMID 22743772, doi:10.1038/nmeth.2019&#160;</td>
		</tr>
		<tr>
			<td>PRIMARY ANTIBODIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tyrosine Hydroxylase Antibody</td>
			<td>Millipore Sigma</td>
			<td>AB1542</td>
			<td>Sheep polyclonal (1:1000 dilution), RRID: AB_90755</td>
		</tr>
		<tr>
			<td>Anti-Tyrosine Hydroxylase Antibody, clone LNC1</td>
			<td>Millipore Sigma</td>
			<td>MAB318</td>
			<td>Mouse monoclonal (1:1000 dilution), RRID: AB_2201528</td>
		</tr>
		<tr>
			<td>Anti-Vesicular Acetylcholine Transporter (VAchT) Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>ABN100</td>
			<td>Goat polyclonal (1:1000 dilution), RRID: AB_2630394</td>
		</tr>
		<tr>
			<td>GFP Antibody</td>
			<td>Novus Biologicals</td>
			<td>NB600-308</td>
			<td>Rabbit polyclonal (1:1000 dilution), RRID: AB_10003058</td>
		</tr>
		<tr>
			<td>Phox2b Antibody (B-11)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-376997</td>
			<td>Mouse monoclonal (1:1000 dilution), RRID: AB_2813765</td>
		</tr>
		<tr>
			<td>SECONDARY ANTIBODIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot)&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-545-152</td>
			<td>Donkey anti-Rabbit (1:400 dilution), RRID: AB_2313584</td>
		</tr>
		<tr>
			<td>AMCA AffiniPure Donkey Anti-Sheep IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td>
			<td>Jackson ImmunoResearch</td>
			<td>713-155-147</td>
			<td>Donkey anti-Sheep (1:400 dilution), RRID: AB_AB_2340725</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Goat IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td>
			<td>Jackson ImmunoResearch</td>
			<td>705-175-147</td>
			<td>Donkey anti-Goat (1:400 dilution), RRID: AB_2340415</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-175-151</td>
			<td>Donkey anti-Mouse (1:400 dilution), RRID: AB_2619678</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Sheep IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td>
			<td>Jackson ImmunoResearch</td>
			<td>713-175-147</td>
			<td>Donkey anti-Sheep (1:400 dilution), RRID: AB_2340730</td>
		</tr>
		<tr>
			<td>RNASCOPE PROBES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Galanin Receptor 1 oligonucleotide probe</td>
			<td>ACDBio</td>
			<td>448821-C1</td>
			<td>targets bp 482 - 1669 (Genebank ref: NM_008082.2)</td>
		</tr>
		<tr>
			<td>Glycine transporter 2 oligonucleotide probe</td>
			<td>ACDBio</td>
			<td>409741-C3</td>
			<td>targets bp 925 - 2153 (Genebank ref: NM_148931.3)</td>
		</tr>
		<tr>
			<td>Phox2b oligonucleotide probe</td>
			<td>ACDBio</td>
			<td>407861-C2</td>
			<td>targets bp 1617 - 2790 (Genebank ref: NM_008888.3)</td>
		</tr>
	</tbody>
</table>

combining multiplex fluorescence in situ hybridization with fluorescent immunohistochemistry on fresh frozen or fixed mouse brain sections

荧光 原位 杂交 （FISH） 是一种分子技术，用于识别细胞内特定 RNA 转录本的存在和空间分布。功能鉴定神经元的神经化学表型分析通常需要使用免疫组织化学 （IHC） 同时使用多种抗体（靶向蛋白）进行标记，并同时优化 原位 杂交（靶向 RNA）。可以实现表征特定神经元的“神经化学特征”，但复杂因素包括需要在组合方法之前验证 FISH 和 IHC 靶标，以及可能在同一组织切片中同时靶向的 RNA 和蛋白质数量有限。
在这里，我们描述了一种使用新鲜冷冻和固定小鼠脑制剂的方案，该方案使用RNAscope FISH检测同一脑切片中的多个mRNA和蛋白质，然后分别进行荧光免疫染色。我们使用组合方法来描述免疫组织化学鉴定的脑干核中低丰度 mRNA（例如甘丙肽受体 1）和高丰度 mRNA（例如甘氨酸转运蛋白 2）的表达模式。
FISH检测下游蛋白质标记的关键考虑因素超出了组织制备和FISH探针标记的优化。例如，我们发现抗体结合和标记特异性可能会受到FISH探针检测中蛋白酶步骤的不利影响。蛋白酶催化肽键的水解裂解，促进 FISH 探针进入细胞，但它们也可能消化随后的 IHC 测定靶向的蛋白质，产生脱靶结合。靶蛋白的亚细胞位置是 FISH 探针检测后促成 IHC 成功的另一个因素。我们观察到，当靶蛋白与膜结合时，IHC特异性得以保留，而靶向细胞质蛋白的IHC需要大量的故障排除。最后，我们发现与新鲜冷冻组织相比，载玻片固定冷冻组织的处理更具挑战性，但是当与RNAscope结合使用时，固定冷冻组织的IHC质量总体上更好。

在这里，我们概述了一种将多重 FISH 与荧光 IHC 相结合的方法，以分别使用新鲜冷冻和多聚甲醛固定组织在小鼠 NTS 中定位 GalR1 和 GlyT2 的 mRNA 表达。图 1 和 图2显示了方法中描述的组织处理、FISH和IHC程序的流程。 表1 总结了每个图中使用的FISH探针和抗体组合。
对照探针与目标探针同时进行常规检测，以确保工作流程?...

Watch this Scientific Journal Video about Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections at JoVE.com

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

该协议描述了一种在新鲜冷冻和固定小鼠脑切片中结合荧光 原 位杂交（FISH）和荧光免疫组织化学（IHC）的方法，目的是实现多标记FISH和荧光IHC信号。IHC 靶向细胞质和膜附着蛋白。

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

该方案结合了荧光RNAscope原位杂交和免疫组化，使用新鲜冷冻或固定的脑制剂。该技术通过展示高灵敏度原位杂交和免疫组织化学方法的组合，实现同一组织标本中 RNA 和蛋白质靶标的空间可视化，从而实现灵活性。该方法适用于脑组织切片的多重标记，有助于研究神经科学中异质神经元群体中mRNA和蛋白质的空间组织和功能意义。该协议主要侧重于新鲜冷冻组织和多聚甲醛固定组织的处理步骤。要开始切片新鲜冷冻的脑组织，请将其固定在预冷的低温恒温器卡盘上，并切割14微米厚的冠状切片。将切片安装到带电的玻璃显微镜载玻片上。将安装的载玻片转移到载玻片盒中。然后将盒子运送到零下 80 摄氏度的干冰冰箱中。对于组织固定，过滤制备的多聚甲醛溶液。冷却至4摄氏度。将安装好的载玻片从零下80摄氏度的冰箱中取出，放在干冰上，立即将其浸入预冷的多聚甲醛溶液中15分钟，对新鲜冷冻的组织进行脱水，以确保载玻片完全干燥。从经心积血固定支架上取出大脑后，使用振动切片机切割 30 微米厚的组织切片。将切割部分储存在冷冻保护剂溶液中。在FISH测定当天，将自由浮动部分转移到含有0.1摩尔PBS的12孔细胞培养板中，并在旋转平台振荡器上以90至100RPM搅拌以洗去冷冻保护剂。然后，使用画笔将切片平放在玻璃显微镜载玻片上，以防止洗涤过程中脱落，并风干至少两个小时。使用疏水屏障笔，在切片周围绘制疏水屏障以容纳FISH试剂。预处理后，将组织在蛋白酶溶液中孵育，并按照制造商的指南与RNAscope探针杂交两小时。然后，进行连续扩增步骤和荧光免疫组化。首先准备一个加湿的避光室，用于孵育载玻片。在水浴中，将 50X 洗涤缓冲液和探头加热至 40 摄氏度 10 分钟。冷却至室温后，从 50X 原液浓度中制备 1 升 1X 洗涤缓冲液。按照文本手稿中的说明制备探针混合物。对于蛋白酶处理，将蛋白酶 3 添加到组织切片上，并在室温下孵育 30 分钟。为了洗掉蛋白酶，在室温下将载玻片轻轻浸入0.1摩尔PBS中，避免切片移位。对于杂交，将探针混合物添加到组织切片上。将其放入加湿的预热室中。然后在 40 摄氏度下孵育两个小时。用洗涤缓冲液冲洗组织部分两次，每次两分钟。对于信号放大，使用滴管瓶，加入扩增溶液以覆盖组织部分并按照文中所述孵育。将封闭溶液添加到组织切片上，并在室温下孵育一小时，以防止非特异性结合。孵育后轻弹载玻片以除去多余的封闭缓冲液。现在，将切片与一抗在 4 摄氏度下孵育过夜。第二天，用 1X TBS 清洗载玻片三次。加入二抗，并在室温下孵育切片两小时。在配备有相机的落射荧光显微镜下检查载玻片。以 20 倍放大倍率采集具有代表性的图像并将其保存为 TIF 文件。在本研究中，通过没有 DAPI 标记来确认组织质量、完整性和无细菌污染。靶向泛素 C、肽基脯氨酰异构酶 B 和 RNA 聚合酶 IIA mRNA 的阳性对照探针的标记证实了 RNA 的完整性。在新鲜冷冻制剂中，为了区分NTS内的GalR1 mRNA阳性神经元，使用了额外的神经化学标志物。膜结合的囊泡转运蛋白被清楚地标记。相比之下，在PHOX2B启动子控制下表达的细胞质蛋白（如酪氨酸羟化酶和GFP）被微弱地观察到并被描述为絮状蛋白，因为它们缺乏清晰的轮廓。GalR1 FISH探针标记细胞质GalR1 mRNA呈点状，观察清晰。为了确认免疫组化质量取决于蛋白质亚细胞定位，在相同的组织切片中用 FISH 平行标记不同的细胞核和细胞质蛋白。细胞质PHOX2B GFP具有絮状外观。而核 PHOX2B 蛋白信号清晰，这证实了与 FISH 联合使用时更高质量的免疫标记。当与FISH联合在固定冰冻切片上进行时，无论亚细胞定位如何，免疫组化都是可靠的。GlyT2 mRNA阳性神经元位于NTS的腹侧，而不是NTS内。GlyT2 mRNA 阳性和 PHOX2B mRNA 阳性神经元不共定位。PHOX2B mRNA 阳性 NTS 神经元的一个亚群具有酪氨酸羟化酶免疫反应性，并且没有一个含有 GlyT2 mRNA。RNAscope能够以单分子分辨率对细胞和组织中的RNA分布进行可视化和分析。它支持对新型 RNA 物种、疾病机制、基因表达模式、神经元多样性和细胞相互作用的研究。

Research

JoVE Journal

Neuroscience

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

将多重荧光原位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究