Name: imaging of podocytic proteins nephrin, actin, and podocin with expansion microscopy
Uploaded: 2021-04-23T14:30:00
Description: Watch this Scientific Journal Video about Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy at JoVE.com

作者要感谢布兰卡·杜夫尼亚克和尼古拉·库尔的出色技术援助。

1. 细胞的分裂和播种
<ol><li>加热无菌杜尔贝科的改良鹰的介质 （DMEM）， 包括 10% 胎儿小牛血清 （FCS）， 无菌磷酸盐缓冲盐水 （PBS） 和无菌肌氨酸到 37 °C. 激活干净的长凳。</li>
	<li>使用无菌钳向每口井添加一个无菌玻璃盖滑（10 毫米），准备一个 6 井板。</li>
	<li>将带有 Cos7 细胞的 10 厘米细胞培养盘放在干净的长凳下。在干净的长凳下，使用真空装置吸气细胞的介质。</li>
	<li>将焦虑的细胞?...

<ol>
	<li>Matsushita, K., 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=Association+of+estimated+glomerular+filtration+rate+and+albuminuria+with+all-cause+and+cardiovascular+mortality+in+general+population+cohorts:+a+collaborative+meta-analysis.">Association of estimated glomerular filtration rate and albuminuria with all-cause and cardiovascular mortality in general population cohorts: a collaborative meta-analysis.</a> Lancet. 375 (9731), 2073-2081 (2010).</li><li>Faul, C., Asanuma, K., Yanagida-Asanuma, E., Kim, K., Mundel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+up:+regulation+of+podocyte+structure+and+function+by+components+of+the+actin+cytoskeleton.">Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton.</a> Trends in Cell Biology. 17 (9), 428-437 (2007).</li><li>Saleem, M. 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=Co-localization+of+nephrin,+podocin,+and+the+actin+cytoskeleton+-+Evidence+for+a+role+in+podocyte+foot+process+formation.">Co-localization of nephrin, podocin, and the actin cytoskeleton - Evidence for a role in podocyte foot process formation.</a> American Journal of Pathology. 161 (4), 1459-1466 (2002).</li><li>Kestila, M., 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=Positionally+cloned+gene+for+a+novel+glomerular+protein+-+nephrin+-+is+mutated+in+congenital+nephrotic+syndrome.">Positionally cloned gene for a novel glomerular protein - nephrin - is mutated in congenital nephrotic syndrome.</a> Molecular Cell. 1 (4), 575-582 (1998).</li><li>Huber, T. B., 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=Podocin-mediated+recruitment+of+nephrin+into+lipid+rafts+is+required+for+efficient+nephrin+signaling.">Podocin-mediated recruitment of nephrin into lipid rafts is required for efficient nephrin signaling.</a> Journal of the American Society of Nephrology. 14, 8 (2003).</li><li>Boute, N., 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=NPHS2,+encoding+the+glomerular+protein+podocin,+is+mutated+in+autosomal+recessive+steroid-resistant+nephrotic+syndrome.">NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.</a> Nature Genetics. 24 (4), 349-354 (2000).</li><li>Galbraith, C. G., Galbraith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+at+a+glance.">Super-resolution microscopy at a glance.</a> Journal of Cell Science. 124 (10), 1607-1611 (2011).</li><li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440 (7086), 935-939 (2006).</li><li>van de Linde, S., 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=Direct+stochastic+optical+reconstruction+microscopy+with+standard+fluorescent+probes.">Direct stochastic optical reconstruction microscopy with standard fluorescent probes.</a> Nature Protocols. 6 (7), 991-1009 (2011).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nature Methods. 3 (10), 793-795 (2006).</li><li>Testa, I., 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=Multicolor+fluorescence+nanoscopy+in+fixed+and+living+cells+by+exciting+conventional+fluorophores+with+a+single+wavelength.">Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength.</a> Biophysical Journal. 99 (8), 2686-2694 (2010).</li><li>Wassie, A. T., Zhao, Y., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+microscopy:+principles+and+uses+in+biological+research.">Expansion microscopy: principles and uses in biological research.</a> Nature Methods. 16 (1), 33-41 (2019).</li><li>Tillberg, P. W., 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=Protein-retention+expansion+microscopy+of+cells+and+tissues+labeled+using+standard+fluorescent+proteins+and+antibodies.">Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies.</a> Nature Biotechnology. 34 (9), 987-992 (2016).</li><li>Truckenbrodt, S., Sommer, C., Rizzoli, S. O., Danzl, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+optimization+in+X10+expansion+microscopy.">A practical guide to optimization in X10 expansion microscopy.</a> Nature Protocols. 14 (3), 832-863 (2019).</li><li>Truckenbrodt, S., 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=X10+expansion+microscopy+enables+25-nm+resolution+on+conventional+microscopes.">X10 expansion microscopy enables 25-nm resolution on conventional microscopes.</a> Embo Reports. 19 (9), (2018).</li><li>Chang, J. B., 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=Iterative+expansion+microscopy.">Iterative expansion microscopy.</a> Nature Methods. 14 (6), 593-599 (2017).</li><li>Chozinski, T. J., 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=nanoscale+optical+imaging+of+mouse+and+human+kidney+via+expansion+microscopy.">nanoscale optical imaging of mouse and human kidney via expansion microscopy.</a> Scientific Reports. 8 (1), 10396 (2018).</li><li>Richter, K. N., 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=Glyoxal+as+an+alternative+fixative+to+formaldehyde+in+immunostaining+and+super-resolution+microscopy.">Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.</a> The EMBO Journal. 37 (1), 139-159 (2018).</li><li>Rinschen, M. M., 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=Quantitative+deep+mapping+of+the+cultured+podocyte+proteome+uncovers+shifts+in+proteostatic+mechanisms+during+differentiation.">Quantitative deep mapping of the cultured podocyte proteome uncovers shifts in proteostatic mechanisms during differentiation.</a> American Journal of Physiology-Cell Physiology. 311 (3), 404-417 (2016).</li><li>Asano, S. M., 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=Expansion+microscopy:+protocols+for+imaging+proteins+and+RNA+in+cells+and+tissues.">Expansion microscopy: protocols for imaging proteins and RNA in cells and tissues.</a> Current Protocols in Cell Biology. 80 (1), 56 (2018).</li><li>Wen, G., 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=Evaluation+of+direct+grafting+strategies+via+trivalent+anchoring+for+enabling+lipid+membrane+and+cytoskeleton+staining+in+expansion+microscopy.">Evaluation of direct grafting strategies via trivalent anchoring for enabling lipid membrane and cytoskeleton staining in expansion microscopy.</a> ACS Nano. 14 (7), 7860-7867 (2020).</li><li>Chen, F., 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=Nanoscale+imaging+of+RNA+with+expansion+microscopy.">Nanoscale imaging of RNA with expansion microscopy.</a> Nature Methods. 13 (8), 679-684 (2016).</li><li>Gambarotto, D., 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=Imaging+cellular+ultrastructures+using+expansion+microscopy+(U-ExM).">Imaging cellular ultrastructures using expansion microscopy (U-ExM).</a> Nature Methods. 16 (1), 71-74 (2019).</li><li>Zhao, Y., 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=Nanoscale+imaging+of+clinical+specimens+using+pathology-optimized+expansion+microscopy.">Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.</a> Nature Biotechnology. 35 (8), 757-764 (2017).</li></ol>

提出的方法使研究者能够可视化细胞蛋白，如波多辛、肾上腺素和细胞骨质成分，例如F-actin。在此协议中，变质的 cos7 细胞用作研究狭缝隔膜蛋白与 F-actin 相互作用的模型。不幸的是，不朽的足细胞系不能表达足够的内源性狭缝隔膜蛋白19。
使用这种方法，细胞蛋白可以使用传统的荧光显微镜以纳米尺度分辨率进行可视化。协议中最关键的步骤是：1） 用 Ac...

白蛋白尿是心血管风险的代孕参数，是球状过滤器1中断的结果。球状过滤器由受精内皮、球状地下室膜和由政治细胞形成的狭缝隔膜组成。主要和次要的足部过程的负细胞包裹在毛细细壁的球状2。足部过程的微妙结构由皮质作用素束保持，皮质作用束也作为多个狭缝隔膜蛋白和其他适配蛋白2的锚。狭缝隔膜的骨干蛋白称为肾素，以同性亲性的方式与对立的 podocyt 的肾素分子相互作用。通过不同的适配器蛋白，肾上腺素与作用细胞球菌2，3有关。肾素编码基因NPHS1的突变导致芬兰4型肾综合征。
肾素的相互作用蛋白之一是波多辛，一种类似发夹的蛋白质的口腔素家族3。波多辛招募肾上腺素脂质筏，并将其链接到行动细胞球菌5。波多辛由NPHS2基因编码。NPHS2中的突变导致类固醇耐肾病综合征6。
为了可视化和共同本地化行为适应蛋白，可以使用免疫荧光技术。不幸的是，光的衍射屏障将传统荧光显微镜的分辨率限制在200-350纳米7。新型显微镜技术，如刺激发射耗竭（STED）8，光激活定位显微镜（PALM）9，随机光学重建显微镜（STORM或dSTORM）或地面状态删除显微镜，然后单个分子返回（GSDIM）9，10，11，使分辨率高达约10纳米。然而，这些超级分辨率技术需要非常昂贵的显微镜，训练有素的人员，因此在许多实验室中是不可用的。
扩展显微镜 （ExM） 是一种新颖而简单的技术，能够使用传统显微镜实现超分辨率成像，并有可能提供给大型研究团体12。在蛋白质保留扩张显微镜（proExM）中，兴趣样本（细胞或组织）是固定的，并沾染了氟磷13。然后，样品中的蛋白质由小分子（6-（丙烯酰）氨基酸、苏奇尼米酰酯、AcX）共同锚定成可膨胀的水凝胶13。通过酶酶K（ProK）的酶消化，蛋白质和氟磷在膨胀13后在凝胶中保持其相对位置。凝胶膨胀后，样品膨胀至4.5倍（体积膨胀90倍），有效横向分辨率约为60-70纳米（300纳米/4.5）。这项技术的修改甚至可以允许10倍的扩展（1000倍体积扩展），使分辨率20-30纳米的传统显微镜14，15，16。
老鼠和人类肾脏的球状结构已经通过ExM17可视化。在本文中，我们提出了一个详细的 ProExM 协议，使用传统的荧光显微镜可视化细胞内 F-actin 和 actin 适配器蛋白 podocin 的超分辨率图像。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrylamide &#62;99%</td>
			<td>Sigma-Aldrich</td>
			<td>A3553-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, Acryloyl-X, SE</td>
			<td>invitrogen</td>
			<td>A-20770</td>
			<td>store up to 4 months</td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Deckgl&#228;ser (cover glasses)</td>
			<td>Engelbrecht</td>
			<td>K12432</td>
			<td>24x32mm #1.0</td>
		</tr>
		<tr>
			<td>Diamont cutter</td>
			<td>VWR</td>
			<td>201-0392</td>
			<td>for cutting the cover slips</td>
		</tr>
		<tr>
			<td>Guanidine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G3272-100G</td>
			<td>8M Stock can be kept at RT</td>
		</tr>
		<tr>
			<td>Marten hair paintbrush</td>
			<td>Leon Hardy</td>
			<td>3 (770)</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Menzel&#34; Deckgl&#228;ser (cover glasses)</td>
			<td>Thermo Fischer&#160;</td>
			<td>15654786</td>
			<td>24x24mm #1.5</td>
		</tr>
		<tr>
			<td>N,N`-Methylenbisacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>M7256-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Objekttr&#228;ger UniMark</td>
			<td>Marienfeld</td>
			<td>703010</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>New England Biolabs</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acrylate</td>
			<td>Sigma-Aldrich</td>
			<td>408220</td>
			<td>check purity&#160;</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining chamber</td>
			<td></td>
			<td></td>
			<td>produced at the university&#39;s workshop</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>ROTH</td>
			<td>2367.1</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well glass bottom plates</td>
			<td>Cellvis</td>
			<td>P06-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Actin-ExM 546&#160;</td>
			<td>chrometra</td>
			<td>non-available</td>
			<td>1:40</td>
		</tr>
		<tr>
			<td>Anti Podocin produced in rabbit</td>
			<td>Sigma</td>
			<td>P-0372-200UL</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Donkey anti guinea-pig CF633</td>
			<td>Sigma</td>
			<td>SAB4600129-50UL</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Goat anti rabbit 488</td>
			<td>Life Technologies</td>
			<td>A11034</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Guinea pig anti nephrin</td>
			<td>Origene</td>
			<td>BP5030</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Visiview</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AXIO Observer Z1</td>
			<td>Zeiss</td>
			<td>non-available</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging of podocytic proteins nephrin, actin, and podocin with expansion microscopy

由球状内皮、球状地下膜和卵石细胞组成的球状过滤器的破坏导致白蛋白尿。多细胞足部过程包含与细胞细胞适配器蛋白（如波多辛）结合的活性素束。这些适配蛋白，如波多辛，将球状狭缝隔膜的骨干，如肾素，与细胞链球菌素连接起来。研究这些蛋白质和其他多囊蛋白的本地化和功能对于理解球状过滤器在健康和疾病中的作用至关重要。所呈现的协议使用户能够在传统显微镜上实现超分辨率成像的细胞中的行为素、波多辛和肾上腺素的可视化。首先，细胞被传统的免疫荧光技术所染色。然后，样品中的所有蛋白质都与膨胀的水凝胶共价锚定。通过用蛋白酶K消化，结构蛋白在最后一步被切开，从而允许凝胶的同位素肿胀。在水中透析样品可使样品膨胀4-4.5倍，样品可以通过传统的荧光显微镜进行成像，潜在分辨率为70纳米。

此 ProExM 协议的概念和时间在图1中进行了描述。在第5天，受感染的细胞被固定并染上荧光抗体，以感兴趣的蛋白质为目标（图1A，B）。在第6天，使用AcX治疗导致所有蛋白质（包括氟磷）（图1A，B）12形成胺组。水凝胶聚合后，这些胺组与水凝胶（第6天）共效结?...

Watch this Scientific Journal Video about Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy at JoVE.com

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

所介绍的方法使荧光标记的细胞蛋白具有扩展显微镜的可视化，从而在传统显微镜上实现 70 nm 的分辨率。

使用扩张显微镜对波多细胞蛋白尼腓林、阿克汀和波多辛的成像

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte ...

这种扩展显微协议使研究者能够在传统的显微镜上用纳米尺度分辨率可视化球状蛋白质。扩展显微镜的主要优点是大型研究社区能够访问显微镜。首先为凝胶室制作垫片。使用钻石刀将一号和 1.5 号玻璃盖切成 5 毫米条纹。将玻璃滑梯放入染色室，并将 1.5 号盖滑条纹放置在玻璃滑梯上，形成 2.5 厘米的正方形。将一滴双蒸馏水放入广场的角落，将玻璃盖滑条粘在彼此之间和玻璃滑梯上。等待约 20 分钟，以便稳定地附加数字 1.5 盖单。每个编号 1.5 盖滑条纹上都有一滴水。将四个头号玻璃盖滑条放在 1.5 号盖滑上。对于凝胶室盖，用石蜡膜包裹盖玻璃，避免薄膜上的任何褶皱或污垢。用于锚定处理，从免疫染色细胞中取出 PBS，并将每口井的锚定缓冲器加入 250 微升，直接固定在玻璃盖滑上。在室温下将盘子孵化三小时。使用盒子将基板保持在黑暗中。孵化后，取下锚定缓冲器，用每口井 1.5 毫升 PBS 清洗盖滑一次。要染色 actin 纤维，解冻与 EXM 兼容的 phalloidin 溶液，并在室温下孵育 45 分钟。同时，使用搅拌装置将丙烯酸钠溶解在双蒸馏水中，并在冰上制备单体溶液。从细胞中取出法罗丁，在室温下用1.5毫升PBS清洗两次。上次洗涤后，将 1.5 毫升 PBS 留在井内。使用钳子和管子将盖玻璃从六个井板上滑落，放入凝胶室。将 APS 添加到凝胶溶液和涡流中。派珀特200微升凝胶溶液在样品上，并谨慎地关闭腔室，避免凝胶内的气泡。将水加入染色室，并在37摄氏度下将染色室孵育至少一小时，使凝胶聚合。将染色室从孵化器中取出。要打开凝胶室盖，在盖子和垫片之间引入剃须刀刀片，并小心地取下盖子。用剃须刀刀片取出隔间，并切断所有额外的凝胶。使用油漆刷分离凝胶的边缘。将滑梯与凝胶和盖玻璃放入装满 PBS 的盘中。然后轻轻摇动凝胶，从凝胶中取出分离的盖玻璃。将滑梯放在凝胶下面，将凝胶连接到滑梯上。在滑梯上使用凝胶，使用剃须刀刀片将凝胶分成小块。轻轻将一块凝胶推入六井板的井中，用玻璃底展开，然后用画笔展开。使用画笔保持凝胶与少量PBS保湿。使用倒置显微镜，对扩展前的图像进行数字光圈低的概述图像。在消化缓冲液中将蛋白酶 K 稀释到每毫升 4 个单位，以制作消化溶液。将消化溶液的 500 微升添加到每口井中，并将凝胶浸入溶液中。让它在室温下消化过夜，盖子关闭，使样品保持在黑暗中。用移液器取出消化液并丢弃。加入一毫升双蒸馏水，在室温下将浸入式凝胶孵化10分钟。取出水，加入一毫升新鲜的双蒸馏水。每 10 分钟继续交换一次水，直到达到膨胀的高原，使凝胶变得光学清晰。从凝胶中取出水，立即开始显微镜检查。使用倒置显微镜，使用低倍率的空气目标来查找处于膨胀前状态的细胞。切换到 40 X 和 63 X 目标以获得更好的分辨率。兴奋的兴趣波长，并通过相机拍摄图像。此 EXM 协议可扩展至多四倍。要确定扩展因子，在扩展前后对细胞进行成像至关重要。锚定不足和均质化可能导致细胞失真和破裂。此处显示了细胞破裂的代表性示例。这种方法可用于研究F-actin和作用适配蛋白的同位素化，如波多辛和肾上腺素。波多辛描绘为绿色，蓝色为行动，肾上腺素为红色。白色区域表示同位化。荧光信号强度和丰度是成功 EXM 的关键。通过 ACX 高效锚定荧光染料对于此协议至关重要。在我们手中溶解的ACX是好长达三到四个月。

Research

JoVE Journal

Biochemistry

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

In Vitro Polymerization of F-actin on Early Endosomes

In Vitro Polymerization of F-actin on Early Endosomes

体外F-肌动蛋白在早期体的聚合

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

用细胞内快速光化学氧化蛋白质对细胞内蛋白质进行特征化

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

使用定量流式细胞术分析荧光标记蛋白与人工磷脂微泡的相互作用

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...