提交人要感谢澳大利亚国家卫生和医学研究理事会项目赠款 APP1103176 支持这项工作。

所有涉及动物组织收集的实验程序都是由纽卡斯尔大学动物保育和伦理委员会批准的。
1. 石蜡嵌入附睾切片的免疫荧光染色 (图1和 2)
<ol><li>在成年小鼠安乐死后, 通过 CO2吸入 (瑞士老鼠, 超过8周), 仔细解剖附睾 (使用手术剪刀和镊子) 没有覆盖结缔组织和脂肪, 浸泡在 Bouin 的固定器解决方案 (> 十倍体积/组织重量) 过夜固定。</li>
	<li>每天用70% 乙醇与2×改变2天, 然后通过分级乙醇 (70%、95% 和 100%) 脱水, 准备入渗和嵌入石蜡块。</li>
	<li>切片的石蜡块在厚度为4-6 µm 和安装在幻灯片上, 准备免疫荧光染色。</li>
	<li>在油烟机中, 脱蜡的附睾石蜡切片添加足够的二甲苯到滑瓶完全浸入组织部分 (3×5分钟每次)。</li>
	<li>水化的组织切片浸泡在分级乙醇溶液稀释纯化 H2O (100% 乙醇5分钟, 100% 乙醇5分钟, 90% 乙醇1分钟, 80% 乙醇1分钟, 70% 乙醇1分钟, 50% 乙醇1分钟)。</li>
	<li>用足够的磷酸盐缓冲盐水 (PBS) 将一个滑动罐中的切片洗涤5分钟, 以完全浸入整个组织部分 (按照这些指示进行后续洗涤)。</li>
	<li>醒酒适当的抗原检索解决方案 (i., 10 毫摩尔柠檬酸钠, 50 毫摩尔/升三 pH 10.5 或替代抗原检索解决方案, 取决于要检测的抗原) 成一个滑动架和微波直到沸腾。将幻灯片浸入此解决方案中, 并将组织切片与热诱导抗原检索条件优化为单个抗体 (见表 1)。 注意: 在抗原检索过程中, 确保幻灯片完全沉浸在抗原检索溶液中。</li>
	<li>从微波炉中取出滑动容器, 冷却至室温。</li>
	<li>用 PBS 冲洗幻灯片, 用一支液态的滑动标记笔在组织切片周围进行跟踪。</li>
	<li>将幻灯片放在一个加湿的容器中 (由容器底部的湿润组织创建), 并应用阻塞解决方案 (以前通过0.45 µm 过滤器过滤的 3% BSA/PBS), 1 小时37摄氏度。</li>
	<li>用 PBS 冲洗一次幻灯片。</li>
	<li>用适当的原抗体稀释到实验性的 1% BSA/PBS 在4°c 过夜 (1:60 用于 anti-DNM1、DNM2 和 DNM3 抗体; 1:100 anti-ATP6V1B1 抗体, 见材料表抗体详细信息)。 注意: 要区分特定于非特异抗体的结合, 必须包括严格的阴性 (i.、二次抗体、原发性抗体 preabsorbed 免疫肽) 和阳性对照24。</li>
	<li>复温在室温下放置30分钟的幻灯片。</li>
	<li>在震动平台 (60 rpm) 上用 PBS 清洗幻灯片3×每10分钟。</li>
	<li>用适当的二级抗体稀释在 1% BSA/PBS (过滤通过0.45 µm 过滤器) 在37°c 为 1 h (1:400 稀释为所有次要抗体, 看见抗体细节的材料表)。 注意: 从这个步骤开始, 将幻灯片容器保持在黑暗中。对于双标记, 选择一个兼容的二次抗体组合 (i. e., 二级抗体必须在不同的物种中提出)。</li>
	<li>在震动平台 (60 rpm) 上用 PBS 清洗幻灯片3×每10分钟。</li>
	<li>Counterstain 碘碘化物 (PI, 7.48 μmol/升) 或 4΄, 6-diamidino-2-苯基吲哚 (DAPI, 4.37 μmol/升) 在室温下的2分钟, 以标记细胞核。</li>
	<li>每两次在震动平台 (60 rpm) 上用 PBS 清洗幻灯片5分钟。</li>
	<li>装上 10% Mowiol 4-88 的部分, 在30% 甘油的溶液中制备0.2 摩尔/升三 (pH 8.5) 和 2.5% 1, 4-杂双环-(2.2. 2)-辛烷值。</li>
	<li>用指甲油封住盖玻片, 将幻灯片存放在4摄氏度以供将来观察。 注意: 建议在准备后尽快执行幻灯片的成像, 以避免过多的荧光损失。</li>
</ol>2从小鼠附睾中分离 Epididymosomes (图 3)
<ol><li>在成年老鼠安乐死以后通过 CO2吸入 (瑞士老鼠8星期老), 灌注他们的血管与 PBS (预热到37°c) 减少附睾组织的血液污染。 注意: 血浆含有不同的外来体种群, 其大小与 epididymosomes25相似。从附睾组织的血液清除效果可以通过检查最初的片段, 一个高度血管化的附睾段位于接近额段 (i. e.,图 1A中的1区)。</li>
	<li>仔细解剖没有上覆脂肪和结缔组织的附睾, 用改良的比格斯、惠顿和 Whittingham 培养基冲洗 (BWW; pH 值 7.4, 渗透压300毫摩尔/千克水26,27), 以减少任何表面电位血液污染。</li>
	<li>涂抹附睾组织去除多余的培养基, 解剖该附睾 (e.,图 1A中的2-5 区), 并转移到含有 BWW 培养基的新鲜培养皿 (35 x 10 毫米)。确保介质数量足以进行最终恢复。 注: 对于6位附睾, 建议使用1.1 毫升的培养基, 以允许恢复900µL, 然后均匀分裂, 并应用在2预准备的梯度之上 (见步骤 2.9)。</li>
	<li>用剃刀刀片将一些小切口插入到头部的组织中。不要切碎组织, 从而避免污染样品与过多胞浆含量。在37°c 的30分钟内, 将含有组织的轻度搅动的钢板孵化, 释放腔内的内容。</li>
	<li>过滤结果悬浮通过70µm 膜去除细胞碎片。</li>
	<li>收集滤液, 并将其与连续离心步骤在4°c, 以提高速度, 以消除细胞碎片 (i., 500 x g, 2000 x g, 4000 x g, 8000 x g, 5 分钟每个;1.7万 x g为20分钟, 最后 1.7万 x g为另外10分钟或直到没有颗粒在离心以后形成)。 注意: 在初始 500 x g离心步骤后对颗粒的颜色进行评估是很重要的, 以确保最小的血液污染存在。丢弃此颗粒显示粉红色着色的任何样品。</li>
	<li>通过稀释密度梯度介质 (包括 60% (w/v) 水 iodixanol), 用0.25 摩尔/iodixanol 蔗糖和10毫摩尔/升三 (pH 7.5) 来制备不连续的梯度 (包括40%、20%、10%、5% 层)。</li>
	<li>在 ultracentrifuge 管 (11 x 35 毫米) 上准备渐变, 每个分数为450µL (图 3)。观察每个分数应用后的渐变, 以确保在加载附睾流体样本之前, 每个层之间成功地形成界面。在使用当天准备每一个渐变, 但是, 在加载前, 附睾腔内流体样品可保存在4摄氏度至2小时。</li>
	<li>小心地添加450µL 的附睾腔内液体悬浮 (对应于从3附睾的头部收集的材料) 在单一梯度之上。</li>
	<li>Ultracentrifuge 梯度在 16万 x g在4°c 为 18 h。 注意: 由于这种离心是在非常高的速度进行, 所有 ultracentrifuge 管必须配对和平衡精确。检查管, 以确保他们没有任何明显的损害, 可能危及他们的完整性。</li>
	<li>轻轻地移除12个相等的分数 (每个由185µL 组成) 从最上层开始, 朝着梯度的底部前进。如果适用 (最多两个渐变), 则池中从每个渐变中恢复的等效分数。 注: 鼠标 epididymosomes 是最丰富的分数 9-1122, 见图 4和讨论。</li>
	<li>在回收和汇集 9-11 分, 稀释成2毫升的 PBS, 并 ultracentrifuge 样品在 10万 x g在4摄氏度 3 h (13 x 56 毫米管) 颗粒的 epididymosomes。 注意: 由于 epididymosome 颗粒很难看到, 确保管的方向被注意到, 因为它们被放置在转子和标记的管, 以表明预期的位置, epididymosome 颗粒。确保每管有足够的容积 (i., 超过其总容量的 50%), 以防止管道坍塌的风险。</li>
	<li>小心地吸入和丢弃上清, 不干扰 epididymosome 颗粒。</li>
	<li>评估 epididymosome 纯度 (图 4)。</li>
	<li>根据下游应用并用重悬 epididymosome 颗粒进入所需介质。例如, BWW 培养基一般用于与精子共孵化的实验, 或者是一个适当的裂解缓冲剂, 用于为附睾蛋白质组通过 SDS 页的分辨率进行准备。</li>
</ol>3. mECap18 细胞的免疫荧光染色
<ol><li>无菌盖玻片的制备 (在细胞培养罩中进行)<ol>
<li>将盖玻片 (12 x 12 毫米) 浸泡在70% 乙醇中10分钟, 在乙醇灯以上高温下干燥消毒。</li>
		<li>冷却的盖玻片在十年代转到12井板块。</li>
		<li>应用无菌聚 l-赖氨酸溶液覆盖盖玻片, 室温下沉淀10分钟。</li>
		<li>放弃聚 l-赖氨酸溶液, 用不育的 H2O 或适当的培养基冲洗盖玻片。</li>
	</ol>
</li>
	<li>制备 mECap18 细胞<ol>
<li>通过 2 x 105 mECap18 细胞的整除数在12井板的每个井包含盖玻片。</li>
		<li>培养细胞与 mECap18 细胞培养基 (DMEM 补充 1% l-谷氨酰胺, 1% 丙酮酸钠, 1% 青霉素/链霉素, 50 μmol/升 5α-androstan-17β-3-oneC-IIIN), 在10% 摄氏度的恒温箱中含有37胎小牛血清 (血清) 5%共2夜。</li>
		<li>一旦细胞坚持盖玻片, 丢弃培养基, 并用 PBS 冲洗两次细胞。</li>
		<li>添加足够数量的4% 多聚甲醛 (粉煤灰) 稀释在 PBS, 以浸泡整个盖玻片和固定在室温下的细胞15分钟。</li>
		<li>丢弃粉煤灰溶液, 在 PBS 中冲洗两次盖玻片。</li>
	</ol>
</li>
	<li>免疫荧光染色<ol>
<li>Permeabilize mECap18 细胞浸泡在0.1% 海卫 X-100 在 PBS 10 分钟。</li>
		<li>用 PBS 冲洗盖玻片。</li>
		<li>阻断 mECap18 细胞与 3% BSA, 并进行 immunolabeling 的细胞使用等效的协议, 为附睾组织切片描述。</li>
	</ol>
</li>
</ol>4. 从条件细胞培养培养基中分离蛋白质
<ol><li>条件细胞培养培养基的收集<ol>
<li>通过整除数 4 x 105 mECap18 细胞在6井板每井与 mECap18 细胞培养基补充10% 个血清 24 h。</li>
		<li>用 mECap18 细胞培养基 (无血清制备) 清洗 mECap18 细胞三次, 以去除残留的血清和任何相关的蛋白质污染物。</li>
		<li>加入1.5 毫升的 mECap18 细胞培养基 (无血清制备), 并在 5% CO2下的37°c 孵化器中与 mECap18 细胞孵育12小时。 注: mECap18 细胞在这一步可以评估不同的目标抗原根据实验设计。</li>
		<li>经过12小时的孵化, 收集细胞培养基和离心机在 2000 x g 10 分钟, 以消除所有细胞碎片。 注: 孵化期可根据实验设计/终点评估, 并考虑细胞对应用治疗的耐受性而改变。建议根据特定的实验方案调整孵化时间, 以确保达到最佳效果。</li>
		<li>通过应用标准的台盼蓝排斥试验评估 mECap18 细胞的生存能力28。丢弃所有细胞存活能力下降到90% 以下的物质, 以消除由死或垂死细胞释放的蛋白质所引入的偏倚。</li>
		<li>将蛋白质从细胞培养基中分离出来, 或保持培养基在-80 摄氏度。</li>
	</ol>
</li>
	<li>蛋白质隔离 (在油烟机中进行)<ol>
<li>添加20% 体积的冷冻100% 乙酸酸到80% 容量的条件细胞培养基, 沉淀从培养的 mECap18 细胞释放的蛋白质。以恒定的混合, 夜间孵化4摄氏度。</li>
		<li>孵化后, 颗粒沉淀蛋白离心 (17, 000 x克, 4 °c 10 分钟)。 注: 由于所需的蛋白质数量有限, 在培养基中可能会使颗粒在离心后不易形象化。因此, 必须在离心前正确定位管, 并注意不要在清除清液时干扰预期颗粒位置。</li>
		<li>在再离心之前, 先用冷冻丙酮将上清液和两次颗粒洗净 (17, 000 x克, 4 °c 10 分钟)。</li>
		<li>在空气干燥之前, 要小心地除去并丢弃上清液, 然后在通风罩内残留丙酮。</li>
		<li>并用重悬蛋白质颗粒在适当的提取缓冲器准备终点分析, 以检测完整的分泌蛋白剖面和/或单个靶蛋白 (e., SDS 页, 免疫印迹法)。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

这些研究纳入了使用 Bouin 的固定附睾组织, 已受到石蜡嵌入和标准切片协议。Bouin 的固定液包括甲醛、苦味酸酸和乙酸的混合物, 每个组分具有特定的互补作用。因此, 甲醛与原胺反应形成蛋白交联, 苦味酸酸慢慢穿透组织形成的盐, 从而使碱性蛋白凝固, 反之, 醋酸迅速穿透组织, 导致凝血核酸。这些组合的性质产生了 Bouin 作为一个固定的选择保存的形态学细节, 它的使用被广泛报道的附睾文献。?...

所有哺乳动物的精子都有可能显示正向渐进运动, 并通过附睾 (雄性额外睾丸导管系统的高度专门化区域) 受精卵子, 这可能用 7-14 天的时间来导航 (取决于物种)1。由于父亲染色质的极度凝结和大部分细胞质的脱落, 伴随着睾丸内精子的 cytodifferentiation, 它们随后的功能成熟完全由它们的相互作用驱动。附睾微环境。这一环境反过来, 由内衬附睾的分泌和吸收活动创造, 并显示一个特殊水平的段段变化1。因此, 在蛋白质合成和分泌方面, 最活跃的部分是位于附睾近端的部分 (即, 额和语料库)2。这项活动反映精子的功能轮廓, 细胞首先开始显示功能能力的特征 (i. e., 渐进动力和绑定到酸可溶性透明糖蛋白的能力) 后, 其通过3。这些功能属性继续发展之前达到最佳水平, 因为精子到达远端附睾段 (马尾), 其中他们储存在一个静止状态, 准备射精。这种精子储存库的形成和维持也与衬里上皮紧密相连, 在马尾中由强吸收活性4、5控制。虽然解剖差异已报告6,7,8, 这种分区分工似乎是一个特点的附睾, 是共享的大多数哺乳动物物种研究至今, 包括我们自己的9,10。事实上, 从临床的角度来看, 附睾功能障碍对男性因素不孕11的病因有重要的贡献, 因此突出了了解这种特殊组织的调节的重要性。
因此, 令人遗憾的是, 我们对附睾生理的理解, 以及调控精子成熟和贮存的顺序阶段的机制, 仍有待完全解决。在促成因素中, 附睾研究的进展受到限制, 这种组织的总体复杂性和对其管腔微环境的调控机制的认识。解剖上, 我们知道, 除了额位, 语料库和马尾段的区别, 附睾可以进一步细分为几个区域 (图 1A), 每隔12间隔, 并以基因/蛋白的离散剖面为特征表达式13,14,15,16,17,18。事实上, 根据对附睾节段基因表达的详细转录分析, 老鼠和大鼠模型中有多达6和9个不同的附睾区, 分别为19、20。这种复杂性大概反映了附睾躯体的组成, 假上皮包括许多不同的细胞类型;每种不同的, 他们的丰度, 分布和分泌/吸收活动的长度。因此, 主细胞是迄今为止最丰富的附睾细胞类型, 构成了所有上皮细胞的80% 以上。因此, 主要细胞负责附睾蛋白生物合成和分泌物的大部分5。与此相反, 作为附睾躯体内第二大细胞类型的透明细胞种群, 主要参与腔内成分的选择性吸收和这种微环境的酸化5。添加另一层复杂性, 雄激素和其他 lumicrine 因素的睾丸起源施加差异控制每一个这些附睾细胞类型取决于他们的定位沿道。
尽管这种复杂性所造成的限制, 仍有重大进展, 以解决附睾功能的机制基础。这些研究的一个关键是应用先进的质谱策略建立广泛的附睾蛋白质组的清单, 并与从这些初步调查中选择的单个蛋白质进行详细分析。这一方法的一个例证是我们最近在老鼠模型21中对 mechanoenzymes DNM 家族的描述。我们最初对 DNM 的兴趣是由它在 endocytotic 过程耦合中的双重作用所推动的。在这些观察的基础上, 我们能够证明 DNM (DNM1-DNM3) 的三规范亚型在小鼠附睾中有高度表达, 并适当定位以在蛋白质分泌和吸收中发挥调节作用21.此外, 我们能够清楚地区分每个 DNM 异构体的基础上, 他们的细胞和亚细胞定位, 从而表明, 他们拥有互补, 而不是多余的活动, 在附睾上皮21。
在这里, 我们描述了用于研究小鼠附睾中 DNM 表达的实验方法, 希望这些信息能在其他附睾蛋白的鉴定中得到更广泛的应用, 从而有助于我们了解男性生殖道这一重要元素的功能。具体来说, 我们描述了在石蜡嵌入附睾切片中靶蛋白免疫荧光标记的稳健方法的发展, 以及随后通过免疫荧光检测这些蛋白质的空间分布。显微镜。我们进一步记录我们最近优化的协议22 , 以隔离和表征 epididymosomes;小的外切状囊泡, 构成附睾分泌剖面的关键元素, 在促进精子成熟23中似乎起着突出的作用。作为补充的方法, 我们还描述了免疫荧光检测的目标蛋白在永生化鼠额附睾上皮 (mECap18) 细胞系和利用这个资源作为一个模型, 以探讨对附睾的调节体外分泌活性。

<table>
	<tbody>
		<tr>
			<td>Dynamin 1 antibody</td>
			<td>Abcam</td>
			<td>ab108458</td>
			<td>Host species: Rabbit, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Dynamin 2 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-6400</td>
			<td>Host species: Goat, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Dynamin 3 antibody</td>
			<td>Proteintech</td>
			<td>14737-1-AP</td>
			<td>Host species: Rabbit, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>ATP6V1B1 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-21206</td>
			<td>Host species: Goat, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>CD9 antibody</td>
			<td>BD Pharmingen</td>
			<td>553758</td>
			<td>Host species: Rat, Isotype: IgG, Class: monoclonal</td>
		</tr>
		<tr>
			<td>Flotillin-1 antibody</td>
			<td>Sigma</td>
			<td>F1180</td>
			<td>Host species: Rabbit, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>ALOX15 antibody</td>
			<td>Abcam</td>
			<td>ab80221</td>
			<td>Host species: Rabbit, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>TUBB antibody</td>
			<td>Santa Cruz</td>
			<td>sc-5274</td>
			<td>Host species: Mouse, Isotype: IgG, Class: monoclonal</td>
		</tr>
		<tr>
			<td>PSMD7 antibody</td>
			<td>Abcam</td>
			<td>ab11436</td>
			<td>Host species: Rabbit, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Anti Rabbit Alexa Fluor 488</td>
			<td>Thermo</td>
			<td>A11008</td>
			<td>Host species: Goat, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Anti Goat Alexa Fluor 488</td>
			<td>Thermo</td>
			<td>A11055</td>
			<td>Host species: Donkey, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Anti Goat Alexa Fluor 594</td>
			<td>Thermo</td>
			<td>A11058</td>
			<td>Host species: Donkey, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Anti Rat Alexa Fluor 594</td>
			<td>Thermo</td>
			<td>A11007</td>
			<td>Host species: Goat, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Anti Rabbit HRP</td>
			<td>Millipore</td>
			<td>DC03L</td>
			<td>Host species: Goat, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Anti Rat HRP</td>
			<td>Millipore</td>
			<td>DC01L</td>
			<td>Host species: Goat, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>Anti Mouse HRP</td>
			<td>Santa Cruz</td>
			<td>sc-2005</td>
			<td>Host species: Goat, Isotype: IgG, Class: polyclonal</td>
		</tr>
		<tr>
			<td>4&#39;, 6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma</td>
			<td>D9564</td>
			<td></td>
		</tr>
		<tr>
			<td>propidium iodide (PI)</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Bovogen</td>
			<td>SFBS-F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Thermo</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#945;-androstan-17&#946;-ol-3-oneC-IIIN</td>
			<td>Sigma</td>
			<td>A8380</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Thermo</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>EMS</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR Chemicals</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Astral</td>
			<td>0497-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>1, 4-diazabicyclo-(2.2.2)-octane</td>
			<td>Sigma</td>
			<td>D2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-gysine</td>
			<td>Sigma</td>
			<td>P4832</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>78787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T6146</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T9159</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Ajax Finechem</td>
			<td>A6-2.5 L GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly (vinyl alcohol)</td>
			<td>Sigma</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Ajax Finechem</td>
			<td>783-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep Density Gradient Medium</td>
			<td>Sigma</td>
			<td>D1556</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Zeiss</td>
			<td>Zeiss Axio Imager A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>BECKMAN COULTER</td>
			<td>Optima Max-XP</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuges</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heracell</td>
			<td>150</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Orbital Shaker</td>
			<td>Ratek</td>
			<td>OM7</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>LG</td>
			<td>MS3840SR /00</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab pH Meter</td>
			<td>MeterLab</td>
			<td>PHM220</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid-repellent slide marker</td>
			<td>Daido Sangyo</td>
			<td>Mini</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Thermo</td>
			<td>586</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>CELLSTAR</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well plate</td>
			<td>CELLSTAR</td>
			<td>665180</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Mikro-Glass</td>
			<td>SF41296PLMK</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filter</td>
			<td>Millox-HV</td>
			<td>SLHV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes Dustfree Paper</td>
			<td>KIMTECH</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tube (2.2 ml, 11 &#215; 35 mm)</td>
			<td>BECKMAN COULTER</td>
			<td>347356</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tube (3.2 ml, 13 &#215; 56 mm)</td>
			<td>BECKMAN COULTER</td>
			<td>362305</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#181;m Nylon</td>
			<td>FALCON</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35 &#215; 10 mm with cams</td>
			<td>SARSTED</td>
			<td>82.1135.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide jar</td>
			<td>TRAJAN</td>
			<td>#23 319 00</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of epididymal protein synthesis and secretion

哺乳动物附睾生成任何内分泌腺体中最复杂的腔内液体之一, 以支持睾丸后成熟和精子的贮存。这种复杂性的产生是由于衬里上皮细胞的分泌和吸收活性的综合作用。在这里, 我们描述的技术, 分析附睾蛋白合成和分泌的重点是模型蛋白家族的动力蛋白 (DNM) mechanoenzymes;具有调节双向膜贩运活动潜力的大型 GTPases。为了研究附睾组织中蛋白质的表达, 我们描述了石蜡嵌入切片中靶蛋白免疫荧光标记的鲁棒方法, 并通过对这些蛋白质的空间分布进行了检测。免疫荧光显微镜。我们还描述了最优化的方法, 以分离和鉴定的外切像囊泡, 称为 epididymosomes, 这是分泌到附睾腔参与细胞间沟通与成熟的精子细胞。作为补充方法, 我们还描述了免疫荧光检测的目标蛋白在 SV40-immortalized 小鼠附睾上皮 (mECap18) 细胞系。此外, 我们还讨论了 mECap18 细胞系作为一种合适的体外模型, 用于研究附睾分泌活动的调节。为此, 我们描述了维持 mECap18 细胞系的培养要求和使用有选择性的药理抑制方案, 它们能够影响其分泌蛋白的分布。后者很容易通过收获的条件培养基, 分泌蛋白的浓度通过乙酸酸/丙酮沉淀和他们随后的分析通过 SDS 页和免疫印迹法。我们认为, 这些联合方法适合于分析替代附睾蛋白靶点, 作为确定它们在精子成熟和/或贮存中功能作用的前奏。

图 1和图 2显示了小鼠附睾 DNM 免疫荧光定位的代表性结果。调查的三 DNM 亚型中的每个都显示不同的定位配置文件。因此, DNM1 的特点是相对温和的弥漫性标记的附睾细胞在整个初始段和额附睾 (图 2A)。相比之下, DNM2 异构体首先检测到在最初的部分细胞的对立的基底和顶端边界附近, 然后被重?...

Watch this Scientific Journal Video about Analysis of Epididymal Protein Synthesis and Secretion at JoVE.com

Analysis of Epididymal Protein Synthesis and Secretion

在这里, 我们报告动力蛋白的免疫荧光定位, 以说明在石蜡嵌入小鼠附睾切片和永生化附睾细胞系 (mECap18) 的蛋白质检测协议。我们还描述了从附睾液和条件细胞培养基中分离分泌蛋白的协议。

附睾蛋白的合成与分泌分析

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation ...

analysis-of-epididymal-protein-synthesis-and-secretion

Research

JoVE Journal

Developmental Biology

9.3K 次观看.  University of Newcastle. 在这里, 我们报告动力蛋白的免疫荧光定位, 以说明在石蜡嵌入小鼠附睾切片和永生化附睾细胞系 (mECap18) 的蛋白质检测协议。我们还描述了从附睾液和条件细胞培养基中分离分泌蛋白的协议。

视频: Analysis of Epididymal Protein Synthesis and Secretion

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

科研

发育生物学

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

心肌细胞DNA的合成和倍性的同时评价:一个方法来帮助心肌细胞再生和营业额的量化

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

血管血管和血管生成的动力学分析体外

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...