Name: isolation, fixation, and immunofluorescence imaging of mouse adrenal glands
Uploaded: 2018-10-02T14:45:00
Description: Watch this Scientific Journal Video about Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands at JoVE.com

我们感谢穆罕默德祖贝尔博士在制定本议定书方面提供的有益建议和技术援助。这项工作得到国家糖尿病和消化和肾脏疾病研究所的支持, 国立卫生研究院 2R01-DK062027 (动力局 H)。

所有方法都是按照在密歇根大学动物使用和护理委员会辖下的体制上批准的议定书进行的。
1. 手术准备
<ol><li>在手术前的前一天, 准备4% 多聚甲醛 (粉煤灰)/磷酸盐缓冲盐水 (PBS)。在冷冻整除数的情况下, 进行解冻, 并贮存在4摄氏度。 注: 4% 粉煤灰不稳定超过48小时。 注意: 粉煤灰是有毒的, 避免接触皮肤和眼睛, 并处理适当的容器。</li>
	<li>手术当天, 用70% 乙醇 (乙醇) 消?...

<ol>
	<li>Brandtzaeg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+power+of+immunohistochemistry+and+immunocytochemistry.">The increasing power of immunohistochemistry and immunocytochemistry.</a> Journal of Immunological Methods. 216 (1-2), 49-67 (1998).</li><li>Matos, L. L., Trufelli, D. C., de Matos, M. G., da Silva Pinhal, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+as+an+important+tool+in+biomarkers+detection+and+clinical+practice.">Immunohistochemistry as an important tool in biomarkers detection and clinical practice.</a> Biomark Insights. 5, 9-20 (2010).</li><li>Coons, A. H., Creech, H. J., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+properties+of+an+antibody+containing+a+fluorescent+group.">Immunological properties of an antibody containing a fluorescent group.</a> Proceedings of the Society for Experimental Biology and Medicine. 47 (2), 200-202 (1941).</li><li>Lerario, A. M., Finco, I., LaPensee, C., Hammer, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Stem/+Progenitor+Cell+Maintenance+in+the+Adrenal+Cortex.">Molecular Mechanisms of Stem/ Progenitor Cell Maintenance in the Adrenal Cortex.</a> Frontiers in Endocrinology. 8, (2017).</li><li>Yates, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+Development,+Maintenance,+and+Disease.">Adrenocortical Development, Maintenance, and Disease.</a> Endocrine Gland Development and Disease. 106, 239-312 (2013).</li><li>Jones, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+the+Mouse+Adrenal+Cortex+with+Special+Reference+to+the+Zona+Reticularis+and+to+Brown+Degeneration,+Together+with+a+Discussion+of+the+Cell+Migration+Theory.">Variation in the Mouse Adrenal Cortex with Special Reference to the Zona Reticularis and to Brown Degeneration, Together with a Discussion of the Cell Migration Theory.</a> Quarterly Journal of Microscopical Science. 89 (1), 53 (1948).</li><li>Sucheston, M. E. C., Samuel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Transient-Zone+in+the+Human+and+Mouse+Adrenal+Gland.">The Transient-Zone in the Human and Mouse Adrenal Gland.</a> The Ohio Journal of Science. 72, 120-126 (1972).</li><li>Guasti, L., Paul, A., Laufer, E., King, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+Sonic+hedgehog+secreting+and+receiving+cells+in+the+developing+and+adult+rat+adrenal+cortex.">Localization of Sonic hedgehog secreting and receiving cells in the developing and adult rat adrenal cortex.</a> Molecular and Cellular Endocrinology. 336 (1-2), 117-122 (2011).</li><li>Pihlajoki, M., Dorner, J., Cochran, R. S., Heikinheimo, M., Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+zonation,+renewal,+and+remodeling.">Adrenocortical zonation, renewal, and remodeling.</a> Frontiers in Endocrinology. 6, 27 (2015).</li><li>Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Droplets+Finally+Get+a+Little+R-E-S-P-E-C-T.">Lipid Droplets Finally Get a Little R-E-S-P-E-C-T.</a> Cell. 139 (5), 855-860 (2009).</li><li>Shen, W. J., Azhar, S., Kraemer, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+and+steroidogenic+cells.">Lipid droplets and steroidogenic cells.</a> Experimental Cell Research. 340 (2), 209-214 (2016).</li><li>Finco, I., LaPensee, C. R., Krill, K. T., Hammer, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+and+steroidogenesis.">Hedgehog signaling and steroidogenesis.</a> Annual Review of Physiology. 77, 105-129 (2015).</li><li>Paul, A., Laufer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+biotin+as+a+marker+of+adrenocortical+cells+with+steroidogenic+potential.">Endogenous biotin as a marker of adrenocortical cells with steroidogenic potential.</a> Molecular and Cellular Endocrinology. 336 (1-2), 133-140 (2011).</li><li>Lowe, J., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stevens &amp; Lowe's Human Histology, 4th Edition. , 263-285 (2015).</li><li>Nagatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+for+human+catecholamine-synthesizing+enzymes.">Genes for human catecholamine-synthesizing enzymes.</a> Neuroscience Research. 12 (2), 315-345 (1991).</li><li>Berthon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+effects+of+Armc5+haploinsufficiency+on+adrenocortical+function.">Age-dependent effects of Armc5 haploinsufficiency on adrenocortical function.</a> Human Molecular Geneticst. 26 (18), 3495-3507 (2017).</li><li>Brown, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+methods+for+immunohistochemistry.">Antigen retrieval methods for immunohistochemistry.</a> Toxicologic Pathology. 26 (6), 830-831 (1998).</li><li>Billinton, N., Knight, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+wood+through+the+trees:+a+review+of+techniques+for+distinguishing+green+fluorescent+protein+from+endogenous+autofluorescence.">Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence.</a> Analytical Biochemistry. 291 (2), 175-197 (2001).</li><li>Hirsch, R. E., San George, R. C., Nagel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+fluorometric+determination+of+the+stable+state+of+aggregation+in+hemoglobins.">Intrinsic fluorometric determination of the stable state of aggregation in hemoglobins.</a> Analytical Biochemistry. 149 (2), 415-420 (1985).</li><li>Whittington, N. C., Wray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+Red+Blood+Cell+Autofluorescence+for+Immunocytochemistry+on+Fixed+Embryonic+Mouse+Tissue.">Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.</a> Current Protocols in Neuroscience. 81, 28 (2017).</li><li>Watson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppressing+autofluorescence+of+erythrocytes.">Suppressing autofluorescence of erythrocytes.</a> Biotechnic &amp; Histochemistry. 86 (3), 207 (2011).</li><li>Epp, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+CLARITY+for+Clearing+Whole-Brain+and+Other+Intact+Organs.">Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs.</a> eNeuro. 2 (3), (2015).</li><li>Erturk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nature Protocols. 7 (11), 1983-1995 (2012).</li></ol>

本协议描述了一种分离小鼠肾上腺的方法以及切片石蜡嵌入小鼠肾上腺的制备和染色。
与我们测试的其他协议相比, 这种免疫荧光协议已被证明适合于我们实验室使用的大多数抗体。然而, 在某些情况下, 它可能需要一些调整, 以改善染色效果。一个可以很容易修改和测试的变量是固定长度。在我们的实验室中, 4% 只粉煤灰的孵化可以从1小时到4小时不等, 而在其他实验室中, 固?...

免疫组化是一种检测组织成分的技术, 使用特定细胞分子的抗体和随后的染色技术检测共轭抗体1。这种免疫组化程序需要特定的固定和处理的组织, 通常是经验主义地确定的特定抗原, 组织和抗体使用2。固定是保持组织的 "原始" 状态的关键, 从而保持完整的细胞和亚胞结构和表达模式。需要进一步的处理和嵌入程序, 以准备组织切片成薄片, 用于组织学研究涉及免疫组化。
染色可以进行显色或荧光检测。显色检测要求利用酶将可溶性基质转化为不溶性的有色产品。虽然这种酶可以与识别抗原的抗体 (主要抗体) 结合, 但它更经常被共轭到识别主要抗体的抗体 (即二级抗体)。这种技术是高度敏感的;由酶反应产生的有色产物是 photostable 的, 只需要 brightfield 显微镜进行成像。然而, 显色染色可能不适合时, 试图想象两个共同本地化的蛋白质, 因为一个颜色的沉积可以掩盖另一个的沉积。在共同染色的情况下, 免疫荧光被证明是更有利的。免疫荧光的出现归因于阿尔伯特库恩斯和同事, 他们开发了一个系统来识别组织抗原与荧光素标记的抗体和可视化他们在切片组织紫外线光3。荧光检测是基于与荧光的抗体共轭的, 在激发后发出光。由于有几个显影在不同波长 (没有或小重叠) 排放, 这种检测方法是研究多种蛋白质的理想选择。
肾上腺是位于肾脏上方的一对配对器官, 其特点是两个 embryologically 不同的成分, 周围有一个间充质胶囊。外肾上腺皮质, 从肾中间胚层, 分泌类固醇激素, 而内部髓质, 来源于神经嵴, 产生儿茶酚胺, 包括肾上腺素, 去甲肾上腺素和多巴酚。肾上腺皮质组织学上和功能上划分三个同心区, 每个区域分泌不同类别的类固醇激素: 外透明球状 (zG) 产生 mineralocorticoids, 调节电解质稳态和血管内容积;中间透明束状带 (zF), 直接在 zG 下方, 分泌糖皮质激素, 通过动员能量储存来调节压力反应, 以增加血糖;和内透明网状 (锆), 合成性类固醇前体 (即,脱氢表雄酮 (DHEAS))4。
在不同种类之间存在肾上腺皮质分带的变化: 例如,毛里求斯缺乏锆。耐热细胞质5, 其独特的产后 X 区为小脂质较差细胞的胎儿皮层残余。X 区在雄性小鼠青春期消失, 在雌性小鼠第一次怀孕后, 或在未繁殖的雌性中逐渐退化6,7。此外, zG 的扭曲和厚度与 zG 附近的外周茎和祖细胞的组织有显著的差异。与其他啮齿动物不同的是, 这只老鼠在 zG 和 zF 之间有一个可见的未分化区 (祖), 它的作用是干细胞区和/或瞬变放大祖细胞区。无论是对大鼠, 还是仅仅是一个更突出的组织细胞群是未知的8,9。
肾上腺皮质的细胞含有脂质滴, 储存胆固醇酯, 作为所有类固醇激素10,11的前兆。 "类固醇激素分泌" 一词定义了从胆固醇生产类固醇激素的过程,通过一系列的酶反应, 涉及类固醇合成因子 1 (SF1) 的活动, 其表达是类固醇合成潜力的标志。在肾上腺, Sf1 表达仅存在于皮质细胞12。一项有趣的研究发现, 类固醇合成电位13的肾上腺皮质细胞内源生物素的表达。虽然这可能是基于生物素/链亲和素染色方法较高背景的原因, 由于检测到内源生物素的抗体共轭与链亲和素, 这一特点也可以用来区分类固醇合成肾上腺内其他人群的细胞,即内皮、囊膜和髓质细胞。
支配由交感神经节前神经元, 肾上腺髓质的特点是嗜碱性颗粒细胞与颗粒细胞质含有肾上腺素和去甲肾上腺素。髓质细胞被命名为 "嗜", 是因为在氧化14后形成褐色色素的儿茶酚胺含量高。酪氨酸羟化酶 (TH) 是催化合成儿茶酚胺的速率限制步骤的酵素, 在肾上腺, 仅表达在髓质15。
在这里, 我们提出了一个小鼠肾上腺的隔离协议, 他们的植入石蜡切片和切片的处理, 并在肾上腺切片上进行免疫荧光染色的方法, 以确定构成的细胞类型肾上腺皮质和髓质。本协议是实验室中用于染色的标准, 用于我们的研究中经常使用的多种抗体。

<table border="0" cellpadding="0" cellspacing="0" style="width:657px;" width="658">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">24-well cell culture plate</td>
			<td style="width:156px;">Nest Biotechnology Co.</td>
			<td style="width:119px;">0412B</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable needles 25Gx5/8&#34;</td>
			<td style="width:156px;">Exel International</td>
			<td style="width:119px;">26403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraplast plus&#160;</td>
			<td style="width:156px;">McCormik scientific</td>
			<td style="width:119px;">39502004</td>
			<td>Paraffin for tissue embedding</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Shandon biopsy cassettes II with attached lid</td>
			<td style="width:156px;">Thermo scientific</td>
			<td style="width:119px;">1001097</td>
			<td>Cassettes for tissue processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High Profile Microtome Blades</td>
			<td style="width:156px;">Accu-Edge</td>
			<td style="width:119px;">4685</td>
			<td>Disposable stainless steel blades</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Peel-a-way disposable plastic tissue embedding molds</td>
			<td style="width:156px;">Polysciences Inc.</td>
			<td style="width:119px;">18986</td>
			<td>Truncated,22mm square top tapered to 12mm bottom</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Superfrost Plus Microscope Slides</td>
			<td style="width:156px;">Fisherbrand</td>
			<td style="width:119px;">12-550-15</td>
			<td>75x25x1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylene</td>
			<td style="width:156px;">Fisher Chemical</td>
			<td style="width:119px;">X5P1GAL&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">200 Proof Ethanol</td>
			<td style="width:156px;">Decon Labs, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Certi-Pad Gauze pads&#160;</td>
			<td style="width:156px;">Certified Safety Mfg, Inc</td>
			<td style="width:119px;">231-210</td>
			<td>3&#34;x3. Sterile latex free gauze pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">M.O.M kit&#160;</td>
			<td style="width:156px;">Vector laboratories</td>
			<td style="width:119px;">BMK-2202</td>
			<td>For detecting mouse primary antibodies on mouse tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KimWipes</td>
			<td style="width:156px;">Kimtech</td>
			<td style="width:119px;">34155</td>
			<td>Wipes 4.4x8.4 inch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Super PAP PEN</td>
			<td style="width:156px;">Invitrogen&#160;</td>
			<td style="width:119px;">00-8899</td>
			<td>Pen to draw on slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope cover glass&#160;</td>
			<td style="width:156px;">Fisherbrand</td>
			<td style="width:119px;">12-544-D</td>
			<td>Size: 22x50x1.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">D9542&#160;</td>
			<td>(Prepared in 20mg/mL stock)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ProLong Gold antifade reagent</td>
			<td style="width:156px;">Molecular Probes</td>
			<td style="width:119px;">P36930</td>
			<td>Mounting agent for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">X-cite series 120Q</td>
			<td style="width:156px;">Lumen Dynamics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coolsnap Myo</td>
			<td style="width:156px;">Photometrics</td>
			<td style="width:119px;"></td>
			<td>Camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optiphot-2&#160;</td>
			<td style="width:156px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microtome</td>
			<td style="width:156px;">Americal Optical</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue embedder</td>
			<td style="width:156px;">Leica</td>
			<td style="width:119px;">EG1150 H&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue processor&#160;</td>
			<td style="width:156px;">Leica</td>
			<td style="width:119px;">ASP300S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal goat serum</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse anti-TH</td>
			<td style="width:156px;">Millipore</td>
			<td style="width:119px;">MAB318</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit anti-SF1</td>
			<td>Ab proteintech group (PTGlabs)</td>
			<td style="width:119px;">&#160;custom made</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa-488 Mouse IgG&#160; raised goat</td>
			<td style="width:156px;">Jackson ImmunoResearch</td>
			<td>115-545-003&#160;</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dylight-549 Rabbit IgG raised goat</td>
			<td style="width:156px;">Jackson ImmunoResearch</td>
			<td>111-505-003</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Citrate acid anhydrous</td>
			<td style="width:156px;">Fisher Chemical</td>
			<td style="width:119px;">A940-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NIS-Elements Basic Research&#160;</td>
			<td style="width:156px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Software for imaging</td>
		</tr>
	</tbody>
</table>

isolation, fixation, and immunofluorescence imaging of mouse adrenal glands

免疫荧光是一种公认的检测组织中抗原的技术, 使用 fluorochrome 共轭抗体, 具有广泛的应用范围。抗原的检测允许对多种细胞类型进行鉴定和鉴定。肾上腺位于肾脏上方, 由一层间充质细胞包裹, 是由两个不同组织组成的内分泌器官, 不同的胚胎起源, 肾中间胚层衍生的外皮质和神经冠的内部髓质。肾上腺皮质分泌类固醇 (即mineralocorticoids、糖皮质激素、性激素), 而肾上腺髓质则产生儿茶酚胺 (即肾上腺素、去甲肾上腺素)。在进行肾上腺研究时, 重要的是要能够区分独特的细胞与不同的功能。在这里, 我们提供了一个在我们的实验室开发的协议, 描述了一系列的顺序步骤, 以获得免疫荧光染色特征的细胞类型的肾上腺。我们首先关注的是小鼠肾上腺的解剖, periadrenal 脂肪的显微切除, 其次是组织的固定、处理和石蜡嵌入。然后我们用旋转切片描述组织块的切片。最后, 我们详细介绍了肾上腺免疫荧光染色的一个协议, 我们已经开发, 以尽量减少非特异性抗体结合和自发荧光, 以达到最佳的信号。

图 1代表了上面描述的整个协议的示意图。肾上腺是从小鼠身上提取的, 在解剖显微镜下切除相邻的脂肪组织, 然后将肾上腺固定在4% 的粉煤灰中。经过这一步, 肾上腺被加工和嵌入石蜡, 并切片与切片, 以削减器官成薄片, 沉积在显微镜幻灯片。切片干燥后, 进行免疫荧光检查, 切片在显微镜下进行成像。
?...

Watch this Scientific Journal Video about Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands at JoVE.com

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

本文提出了一种从小鼠中分离肾上腺的方法, 修复组织, 切片, 并进行免疫荧光染色。

小鼠肾上腺的分离、固定和免疫荧光成像

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

免疫荧光是一种基本方法，可以帮助回答肾上腺领域有关肾上腺区、细胞特性和功能的关键问题。这项技术的主要优点是，它允许研究多种蛋白质，同时保持肾上腺组织的形态。要去除粘液肾上腺，切开周围的脂肪组织，将腺体放在含有新鲜 PBS 的 24 多井板的一个井中。在解剖显微镜下将腺体转移到培养皿或玻璃支撑上，并使用两根 25 量针去除相邻的脂肪组织。当所有的脂肪都去除后，在4摄氏度下将组织用4%的半甲醛固定，摇动两个小时。在孵育结束时，在4摄氏度下用3个15分钟洗涤腺体，每次洗涤一至两毫升新鲜PBS。最后一次洗涤后，在四摄氏度的两次连续乙醇浸入中脱水腺体，每次孵育摇动两小时。要准备肾上腺组织进行加工，请用纱布包裹腺体，并将肾上腺包裹在组织嵌入的盒式磁带中。然后，将盒式磁带放在装满 70% 乙醇的罐子中，储存四摄氏度，直到加工。要处理组织，请将盒式磁带插入组织处理器中进行处理，如表中所示。在程序结束时，将盒式磁带转移到一个嵌入站，里面装满了融化的 65 度石蜡，并使用一对钳子来解包纱布。轻轻地将肾上腺转移到嵌入模具底部中心的适当位置，将融化的石蜡倒入腺体。然后，小心地将嵌入模具移动到冷却盘上，以方便石蜡硬化。在旋转微原子进行剖切之前，先给一系列显微镜幻灯片贴上标签，然后将幻灯片放在 37 摄氏度的幻灯片加热器上。将大约 450 微升的高压处理类型一个超纯水分配到每个幻灯片上，将微原子的截面厚度设置为 5 微米。将嵌入的组织加载到切割块上，并降低石蜡块，直到样品与微切刀边缘处于面部水平。以稳定的节奏旋转手轮，执行初始修剪以去除石蜡并暴露肾上腺组织。然后，通过肾上腺的端部获取五微米分段，使用钳子将每个部分转移到水滴中，在收集时将其转移到预先标记的幻灯片上。通过显微镜确认部分中是否存在组织后，将热板上的幻灯片干燥约两个小时，并在 37 摄氏度的滑动托盘上至少烘烤一夜，直到看不到更多的水。干燥后，将幻灯片存放在室温下滑梯盒中。对于免疫荧光抗原检索，用两个五分钟100%二甲苯浸入将幻灯片去质，然后进行降乙醇和水浸水系列的补液。浸入水后，将滑梯转移到金属滑架上，将滑轨放在沸腾的0.01摩尔柠檬酸盐烧杯中，放在热板上10分钟。在孵育结束时，从热板中取出烧杯，让它冷却20分钟，注意所有肾上腺部分仍然覆盖着缓冲液。当烧杯冷却后，将幻灯片转移到科普林罐中，用柔和的摇动在新鲜的 PBS 中洗涤 3 个 10 分钟。最后一次洗涤后，小心擦拭每张幻灯片的表面，而不损坏组织部分，并使用疏水标记在每个部分周围绕一个圆圈。将幻灯片放在加湿室中，并快速向每个部分添加至少 50 微升的阻尼溶液，在室温下孵育一小时。在阻塞孵育结束时，用新鲜 PBS 将幻灯片洗三次，每次摇动五分钟，然后将幻灯片返回腔室。接下来，将感兴趣的原抗体鸡尾酒添加到每个部分，在摄氏四度下进行过夜孵育。第二天早上，每次洗涤用三个15分钟的新鲜PBS清洗样品，并用适当的二次抗体溶液标记幻灯片，防止光线一小时。在孵化结束时，在防止光线的摇杆上用三个15分钟的洗涤洗涤样品，在上次洗涤后在室温下用DAPI标记各部分的核7分钟。经过三个五分钟的PBS用摇摆洗涤后，沿每个样品一个盖玻璃的表面添加60微升适合免疫荧光的安装剂，然后轻轻按压每个滑动组织部分侧下到盖玻璃上，注意避免气泡。然后，将每个幻灯片平放在深色容器中，在室温下固化至少 24 小时，然后进行成像。在脂肪去除过程中，至关重要的是不要让肾上腺干涸，因为脱水会损害组织结构。成功去除周围脂肪组织后，肾上腺应易于检测，无需额外的可见脂肪。肾上腺部分的免疫染色，如证明揭示了核类固醇因素一染色的肾上腺细胞，酪氨酸羟基酶染色的乳腺细胞，DAPI染色的非类固醇细胞的外胶囊核，并共同染色与肾上腺和乳腺细胞。尝试此过程时，重要的是要记住，鼠标前科很小，它们的位置有些变化，使得它们的检测困难。因此，在解剖过程中要清楚地了解该区域。这些程序是研究体内肾上腺的基本技术。之后，可以执行额外的免疫污染方法，以满足特定的科学需要。经过开发，免疫污染为生物医学领域的研究人员探索原位蛋白表达铺平了道路，这是许多学科和领域的有用工具。

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Immunology and Infection

Unit 1

The Endocrine System

SCIENTISTS IN ACTION

Isolation of Mouse Peritoneal Cavity Cells

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

科研

免疫与感染

Isolation of Mouse Lung Dendritic Cells

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Intravital Imaging of the Mouse Popliteal Lymph Node

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

小鼠特拉切斯刷细胞的分离与定量评价

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

从人类唾液腺中分离唾液表皮细胞,作为脂质层或单层体进行体外生长

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

小鼠中人类胸膜皮质瘤的PET/CT的植入与监测

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...