Name: microdissection and dissociation of the murine oviduct: individual segment identification and single cell isolation
Uploaded: 2021-11-04T13:00:00
Description: Watch this Scientific Journal Video about Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation at JoVE.com

这项工作得到了国防部对AMW突破奖（BCRP W81XWH-14-1-0425）的部分支持。KCR部分得到了校内奖学金的支持：Pease癌症博士预科奖学金和Mary Galvin Burden生物医学科学博士前奖学金以及加州大学河滨分校，校内奖：研究生理事会奖学金委员会论文研究补助金和研究生部论文年度计划奖。作者感谢Gillian M. Wright和Alyssa M. Kumari在这种方法的早期故障排除方面的帮助。

所有动物处理和程序均由加州大学河滨分校机构动物护理和使用委员会批准，并符合美国实验动物护理协会，美国农业部和美国国立卫生研究院的指南。所述方法利用C57BL/6成年雌性小鼠。所有动物在组织收获前都通过斩首实施了安乐死。
注：该方案的概述，使用蓝色染料来帮助有效解剖和解开输卵管，如第一图所示（图1）。
<p class="jove_content" fo:keep...

<ol>
	<li>Stewart, C. A., et al., Kubiak, J. Z., 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=.">.</a> Mouse oviduct developmemt in Mouse Development: From Oocyte to Stem Cells. , 247-262 (2012).</li><li>Ford, M. 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=Oviduct+epithelial+cells+constitute+two+developmentally+distinct+lineages+that+are+spatially+separated+along+the+distal-proximal+axis.">Oviduct epithelial cells constitute two developmentally distinct lineages that are spatially separated along the distal-proximal axis.</a> Cell Reports. 36 (10), 109677 (2021).</li><li>Harwalkar, 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=Anatomical+and+cellular+heterogeneity+in+the+mouse+oviduct-its+potential+roles+in+reproduction+and+preimplantation+development.">Anatomical and cellular heterogeneity in the mouse oviduct-its potential roles in reproduction and preimplantation development.</a> Biology of Reproduction. 104 (6), 1249-1261 (2021).</li><li>Agduhr, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+structure+and+development+of+the+bursa+ovarica+and+the+tuba+uterina+in+the+mouse.">Studies on the structure and development of the bursa ovarica and the tuba uterina in the mouse.</a> Acta Zoologica. 8 (1), 1 (1927).</li><li>Pulkkinen, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviductal+function+is+critical+for+very+early+human+life.">Oviductal function is critical for very early human life.</a> Annals of Medicine. 27 (3), 307-310 (1995).</li><li>Kindelberger, D. 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=Intraepithelial+carcinoma+of+the+fimbria+and+pelvic+serous+carcinoma:+Evidence+for+a+causal+relationship.">Intraepithelial carcinoma of the fimbria and pelvic serous carcinoma: Evidence for a causal relationship.</a> American Journal of Surgical Pathology. 31 (2), 161-169 (2007).</li><li>Ghosh, A., Syed, S. M., Tanwar, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genetic+cell+lineage+tracing+reveals+that+oviductal+secretory+cells+self-renew+and+give+rise+to+ciliated+cells.">In vivo genetic cell lineage tracing reveals that oviductal secretory cells self-renew and give rise to ciliated cells.</a> Development. 144 (17), 3031-3041 (2017).</li><li>Lee, 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=A+candidate+precursor+to+serous+carcinoma+that+originates+in+the+distal+fallopian+tube.">A candidate precursor to serous carcinoma that originates in the distal fallopian tube.</a> Journal of Pathology. 211 (1), 26-35 (2007).</li><li>Kim, 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=High-grade+serous+ovarian+cancer+arises+from+fallopian+tube+in+a+mouse+model.">High-grade serous ovarian cancer arises from fallopian tube in a mouse model.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (10), 3921-3926 (2012).</li><li>Perets, 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=Transformation+of+the+fallopian+tube+secretory+epithelium+leads+to+high-grade+serous+ovarian+cancer+in+Brca;Tp53;Pten+models.">Transformation of the fallopian tube secretory epithelium leads to high-grade serous ovarian cancer in Brca;Tp53;Pten models.</a> Cancer Cell. 24 (6), 751-765 (2013).</li><li>Sherman-Baust, C. 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=A+genetically+engineered+ovarian+cancer+mouse+model+based+on+fallopian+tube+transformation+mimics+human+high-grade+serous+carcinoma+development.">A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.</a> Journal of Pathology. 233 (3), 228-237 (2014).</li><li>Zhai, Y. L., 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=High-grade+serous+carcinomas+arise+in+the+mouse+oviduct+via+defects+linked+to+the+human+disease.">High-grade serous carcinomas arise in the mouse oviduct via defects linked to the human disease.</a> Journal of Pathology. 243 (1), 16-25 (2017).</li><li>Karthikeyan, 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=Prolactin+signaling+drives+tumorigenesis+in+human+high+grade+serous+ovarian+cancer+cells+and+in+a+spontaneous+fallopian+tube+derived+model.">Prolactin signaling drives tumorigenesis in human high grade serous ovarian cancer cells and in a spontaneous fallopian tube derived model.</a> Cancer Letters. 433, 221-231 (2018).</li><li>Shao, 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=Differences+in+prolactin+receptor+(PRLR)+in+mouse+and+human+fallopian+tubes:+Evidence+for+multiple+regulatory+mechanisms+controlling+PRLR+isoform+expression+in+mice.">Differences in prolactin receptor (PRLR) in mouse and human fallopian tubes: Evidence for multiple regulatory mechanisms controlling PRLR isoform expression in mice.</a> Biology of Reproduction. 79 (4), 748-757 (2008).</li><li>Alwosaibai, 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=PAX2+maintains+the+differentiation+of+mouse+oviductal+epithelium+and+inhibits+the+transition+to+a+stem+cell-like+state.">PAX2 maintains the differentiation of mouse oviductal epithelium and inhibits the transition to a stem cell-like state.</a> Oncotarget. 8 (44), 76881-76897 (2017).</li><li>Peri, L. E., 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=A+novel+class+of+interstitial+cells+in+the+mouse+and+monkey+female+reproductive+tracts.">A novel class of interstitial cells in the mouse and monkey female reproductive tracts.</a> Biology of Reproduction. 92 (4), 102 (2015).</li><li>L&#245;hmussaar, 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=Assessing+the+origin+of+high-grade+serous+ovarian+cancer+using+CRISPR-modification+of+mouse+organoids.">Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids.</a> Nature Communications. 11 (1), 2660 (2020).</li><li>Chen, 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=An+air-liquid+interphase+approach+for+modeling+the+early+embryo-maternal+contact+zone.">An air-liquid interphase approach for modeling the early embryo-maternal contact zone.</a> Scientific Reports. 7, 42298 (2017).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> Journal of Visualized Experiments: JoVE. (45), e2565 (2010).</li><li>Schroeder, 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=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Molecular Biology. 7, 3 (2006).</li><li>McGlade, E. 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=Cell-type+specific+analysis+of+physiological+action+of+estrogen+in+mouse+oviducts.">Cell-type specific analysis of physiological action of estrogen in mouse oviducts.</a> The FASEB Journal. 35 (5), 21563 (2021).</li></ol>

输卵管的三个节段在组织学上，形态学上和功能上是不同的1，2，3。上皮从输卵管的一端到另一端变化很大。纤毛细胞在腓肠/腹肌末端占主导地位，而分泌细胞在峡部区域占主导地位1。虽然这种总体梯度已经被认识到了一段时间，但最近的工作已经发现了输卵管节段之间的更多区别。因此，虽然纤毛细胞在?...

小鼠输卵管在功能和形态上与人类输卵管相似1。两者都由假分层的纤毛上皮组成，由两个历史上描述的上皮常驻细胞组成：纤毛细胞和分泌细胞1，2。输卵管有三个经典公认的部分：腹腔，壶腹和峡部。在最近的一项研究中，Harwalkar等人。3 研究了输卵管形态和基因表达，导致将常驻上皮细胞的分类扩展到七个不同的群体。此外，他们建立了壶腹 - 地峡连接处作为输卵管3的独特部分。本文描述的方法侧重于腹足、壶腹和峡部，可以很容易地扩展到包括壶腹-峡部交界处2，3。腹股沟区域包含输卵管的开口，包括腓肠区域以及近端茎。向子宫移动，接下来是壶腹，然后是峡部。纤毛细胞在该区域的远端、卵巢近端或足底最为突出，而分泌细胞在近端或峡部最为突出1。与人类输卵管不同，小鼠输卵管是由腹膜宽韧带的延伸中脉支撑的盘绕结构1，4。此外，小鼠输卵管被包裹在滑囊中，这增加了卵母细胞转移到输卵管中的可能性4。壶腹被确定为受精的位置，发育中的胚胎在进入子宫之前从该位置进入地峡5。输卵管节段直径为200-400μm，较长的壶腹和峡部区域的长度约为0.5-1.0厘米4。输卵管在发情周期期间膨胀，腹扩和腹股沟比峡更易扩张1。
细胞（尤其是分泌细胞）的过度增殖是盆腔中发现的浆液性肿瘤的前体病变的特征6。这些前体浆液性上皮内病变仅在腓骨区域出现在输卵管上皮中;目前尚不清楚为什么病变形成仅限于该区域，其中通常主要细胞类型是纤毛的，而不是分泌的2，7，8。正常生理功能的区域性以及对卵巢癌输卵管起源的兴趣增加9，10，11，12，13，强调了单独评估输卵管段的重要性。
这里描述的方法详细介绍了单独输卵管片段的收集，用于随后对片段特异性基因表达和解离细胞的功能进行下游分析。传统上，许多组织按照苯酚：氯仿法或柱上完全提取法进行全RNA提取;然而，我们发现RNA质量得以维持，同时使用所述的组合方法产生了足够的产量。利用这种方法，可以处理输卵管的非常小的功能段以进行下游分析，而不是将输卵管作为一个整体进行研究，这可以掩盖代表不同段的结果14。
分离的小鼠输卵管细胞很少通过流式细胞术进行研究，很可能是由于该组织的细胞产量有限。克服这个问题的一种方法是解离细胞，在培养物中培养它们，然后在 体外 刺激再分化，以获得适当的细胞数用于下游细胞分析15，16，17，18。这种方法的局限性是培养物中 离体 时间和微环境的改变，这两者都可能改变基因表达。还有一种假设是，形态学上的再分化具有与完整动物相同的转录和蛋白质组学特征。目前的解离方法旨在实现异质性输卵细胞群中最高数量的上皮细胞，同时保持单细胞分化。此外，大多数非酶的方法可能会限制细胞表面蛋白的损失。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mm Stainless steel bead mix</td>
			<td>Next Advance</td>
			<td>SSB05</td>
			<td>Mix 1:1 with 1.4 mm SSB14B, sterilized</td>
		</tr>
		<tr>
			<td>1.4 mm Stainless steel bead mix</td>
			<td>Next Advance</td>
			<td>SSB14B</td>
			<td>Mix 1:1 with 0.5 mm SSB05, sterilized</td>
		</tr>
		<tr>
			<td>1X Dulbecco&#39;s Phosphate Buffered Saline A, pH 7.4 (DPBS)</td>
			<td>Gibco</td>
			<td>21600-010</td>
			<td>Cold, sterile</td>
		</tr>
		<tr>
			<td>25G needle</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm sterile petri dishes</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell meshes</td>
			<td>Fisherbrand</td>
			<td>22-636-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Eukaryote Total RNA 6000 Pico Chip kit</td>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Bullet Blender Tissue Homogenizer</td>
			<td>Next Advance</td>
			<td>BBY24M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanlyzer</td>
			<td>Agilent 2100 Bioanalyzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold plate/pack/surface of choice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Kept at -20C for dissection</td>
		</tr>
		<tr>
			<td>Dental wax</td>
			<td>Polysciences Inc.</td>
			<td>403</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/ Ham&#8217;s F12</td>
			<td>Corning</td>
			<td>10-090-CV</td>
			<td>Prepare dissection medium: DMEM/ Ham&#8217;s F12, 10% FBS, 25 mM Hepes, 1% Pen-Strep</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-015CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine point forceps of choice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>G712b</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-21422</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11001</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H-3784</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>Cell Signaling Technologies</td>
			<td>4082S</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Inverted compound microscope</td>
			<td>Keyence BZ-X700</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse Occludin</td>
			<td>Invitrogen</td>
			<td>33-1500</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Non-enzymatic dissociation buffer</td>
			<td>N/A</td>
			<td></td>
			<td>5 mM EDTA, 1 g/L glucose, 0.4% BSA, 1X DPBS</td>
		</tr>
		<tr>
			<td>Nylon macro-mesh 1 mm x 1 mm</td>
			<td>Thomas Scientific</td>
			<td>1210U04</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P-6148</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>MP Biomedicals</td>
			<td>10220-718</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent</td>
			<td>Cell Signaling Technologies</td>
			<td>9071S</td>
			<td>Immunocytochemical validation images: antifade mounting medium</td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma Aldrich</td>
			<td>10165921001</td>
			<td>Prepare pronase digestion medium: 0.15% Pronase in DMEM/Ham&#39;s F12, sterile</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Roche</td>
			<td>11 348 639 001</td>
			<td>Viability validation images</td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse Acetylated-Tubulin</td>
			<td>Abcam</td>
			<td>ab179484</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>Utilized for on-column purification in text.</td>
		</tr>
		<tr>
			<td>Spring form microdissection scissors</td>
			<td>Roboz Surgical</td>
			<td>RS-5610</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 3 mL bulb pipettors</td>
			<td>Globe Scientific</td>
			<td>137135</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue</td>
			<td>Alfa Aesar</td>
			<td>J66015</td>
			<td>Prepare toluidine blue solution: 1% in 1X DPBS, sterile</td>
		</tr>
		<tr>
			<td>Tris-Buffered Saline-Tween (TBST)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Immunocytochemical validation images; 0.1 N NaCl, 10 mM Tris-Cl pH 7.5, 1% Tween 20</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Mallinckrodt Inc.</td>
			<td>3555</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Trizol RNA Extraction Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>Referred to as RNA extraction reagent. Nucelase-free water, chloroform and isoproponal are required in supplement of performing Trizol extraction per manufacturer&#39;s guidelines</td>
		</tr>
	</tbody>
</table>

microdissection and dissociation of the murine oviduct: individual segment identification and single cell isolation

小鼠模型系统在疾病过程分析方面是无与伦比的，因为它们具有遗传可操纵性和实验性治疗的低成本。然而，由于它们的体型小，一些结构，如直径为200-400μm的输卵管，已被证明相对难以研究，除非通过免疫组织化学。最近，免疫组织化学研究揭示了输卵管段比以前认识到的更复杂的差异;因此，输卵管被分为四个功能段，具有七种不同上皮细胞类型的不同比例。上皮细胞类型的不同胚胎起源和比例可能使这四个功能区域对疾病有不同的易感性。例如，浆液性上皮内癌的前体病变来自小鼠模型中的内底和人类输卵管中的相应腓肠区域。这里描述的方案详细介绍了一种显微解剖方法，以产生下游分析所需的足够量和纯度的RNA，例如逆转录定量PCR（RT-qPCR）和RNA测序（RNAseq）。还描述了一种大部分非酶解组织解离方法，适用于流式细胞术或单细胞RNAseq分析的完全分化的输卵管细胞。所描述的方法将促进在生殖，生育，癌症和免疫学领域利用小鼠输卵管的进一步研究。

所描述的解离方案每只小鼠产生100，000-120，000个细胞，两个输卵管池化。该方法足够温和，可以保持多纤毛细胞边界完整，从而可以区分多纤毛细胞和分泌细胞，并验证消化方法是否足够温和以防止去分化。图5中的代表性免疫荧光图像显示了步骤4.2.1之后的小细胞团块，在4%多聚甲醛（PFA）/ DPBS中固定3分钟，用1%牛血清白蛋白（BSA）/Tris缓冲盐水补间（BTBST）洗涤并在悬浮液...

Watch this Scientific Journal Video about Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation at JoVE.com

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

本文提出了一种小鼠输卵管显微解剖方法，该方法允许在保持RNA完整性的同时收集单个片段。此外，描述了非酶促卵管细胞解离过程。这些方法适用于功能不同的输卵管节段和解离的输卵管细胞的后续基因和蛋白质分析。

小鼠输卵管的显微解剖和解离：个体片段鉴定和单细胞分离

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

不同的输卵管节段具有不同的生理功能，并且对病理变化具有不同的易感性。在非常小的小鼠输卵管中鉴定不同的片段具有挑战性，通过组织解离产生良好分化的细胞的良好产量也是如此。在本视频中，我们将演示如何解开小鼠输卵管的线圈，识别不同的片段以便可以单独分析它们，最后，如何产生相对高产量的分化解离细胞。更好地了解壶腹特有的环境可以改善体外受精技术。此外，通过将内底与其他部分进行比较，我们可以更好地了解内底的恶性转化倾向。单细胞解离程序从输卵管中释放基质细胞以及上皮细胞。我们的方法还提供了单独分析免疫，平滑肌和上皮细胞对输卵管生理学和病理学的贡献的机会。起初，在解剖显微镜下工作可能很困难。我建议使用更大的管子（如子宫）进行练习，以确保您的工具足以进行输卵管显微切除术。首先将一块牙蜡贴在带有粘合剂的培养皿上，然后让它变干。然后用70%乙醇消毒盘子。表面现在已准备好进行解剖。准备解剖时，在解剖显微镜下将含有牙蜡的培养皿固定在冷平台上。用70%乙醇消毒动物的腹侧表面。然后用无菌解剖剪刀打开腹腔和盆腔。将胃肠道移动到一侧以定位背侧双角子宫。沿着每个子宫角向喙方向移动，以定位包裹在肾脏正下方的子宫脂肪垫中的每个输卵管和卵巢。然后用卵巢切除两个侧输卵管和仍然用解剖剪刀连接的远端子宫角的一部分。切开每个子宫外侧角，大约一到两厘米的角仍然附着在盘绕的输卵管和卵巢上。立即将组织浸没在两毫升冷解剖介质中，然后用无菌的25号针将子宫组织贴在牙蜡上，以确保组织进行解剖。清洁并解剖卵巢脂肪垫和结缔组织，以清晰地观察输卵管。使用一毫升注射器分配一到两滴无菌的1%甲苯胺蓝色染料溶液，并孵育30秒至一分钟。用冷DPBS冲洗，然后除去所有液体。轻轻地将卵巢从盘绕的输卵管中拉出。在宽韧带的滑囊膜处切开，以从输卵管中取出卵巢，而不会影响输卵管组织。定位输卵管的远端腓肠末端，该端位于子宫输卵管连接处的侧侧。轻轻拉动管子，用弹簧形微剪刀切开中胚层，以解开输卵管。切开管子以产生心底区域。然后通过在第二圈和第三圈之间切割来切除壶腹区域。最后，将剩余部分切到子宫输卵管连接处，这是峡部区域。立即在液氮中快速冷冻解剖的组织片段，然后向组织中加入200微升冷RNA提取试剂和一对一的均质微球混合物进行RNA分离。从腓骨区域开始，用弹簧剪刀纵向切开管子，并使用镊子作为杠杆来暴露腔内上皮。将切开的部分和剩余的输卵管切成大约一到两毫米的段。然后向组织中加入5毫升温热的非酶解解缓冲液，并在37摄氏度下孵育。此处显示了闭塞素、细胞核和乙酰化微管蛋白阳性细胞簇的荧光和相差图像。红色表示闭塞素，蓝色表示细胞核，绿色表示乙酰化微管蛋白。纤毛顶端边界在整个方案中保持完整，并承受对单个细胞的进一步消化。在这些具有代表性的图像中，显示了合并的红色、蓝色、相衬和绿色通道。经过短暂的蛋白酶孵育后，大多数细胞是单一的。碘化丙啶染色在明场、红色通道 PI 阳性和合并中显示出约 93% 的活力。合并时的插图显示单个解离单元格上的完整纤毛边框。这样的细胞具有活跃的跳动纤毛。通过微量分光光度计和生物分析仪评估整个RNA的产量和质量。结果表明，该方法每段产生800至1， 200纳克RNA，但输液管样品除外，其中可能需要从两只动物中汇集以获得下游测定的适当产量。所呈现的内脑袋结果来自两只动物，共四个腹足区。该协议成功地实现了使用微量分光光度计和生物分析仪分析的纯RNA。样品的吸收比为260 x 280，介于1.8和2之间，是纯RNA核苷酸物种的典型特征。生物分析样品显示出高RNA完整性，评估的RNA完整性值高于7，适合下游表达和测序分析。请记住保留一到两厘米的子宫角，以将输卵管系统固定在解剖平台上。此外，使用甲苯胺蓝色染料有助于输卵管的快速开卷。开卷速度越快，保存效果越好，RNA降解量越少。该方法足以获得适当质量的RNA，用于允许单个片段测序的RNA测序。细胞解离适用于单细胞测序或流式细胞术，允许更精确的片段和细胞分析。该方法将有助于将视野从全管分析中移开，从而掩盖片段或单细胞水平的差异。

Research

JoVE Journal

Developmental Biology

Murine Dermal Fibroblast Isolation by FACS

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

科研

发育生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

从舌头上皮和梅森奇姆/胚胎日12.5和8周大的老鼠的结缔细胞分离

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

从小鼠和人类牙齿中快速分离单个细胞

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

流式细胞术从小鼠组织中鉴定和分离爆破形成单元和集落形成单元红系祖细胞

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...