冈 比亚按蚊 幼虫唾液腺的解剖和免疫染色
Summary
成年蚊子唾液腺（SG）是将所有蚊子传播的病原体（包括病毒和寄生虫）传播给人类宿主所必需的。该视频演示了从幼虫（L4）阶段冈 比亚按蚊 中有效分离SG以及L4 SGs的制备以进行进一步分析。
Abstract
蚊子唾液腺（SGs）是传播昆虫传播病原体的必要门户器官。致病因子，包括病毒和引起疟疾的疟原虫寄生虫，积聚在SG细胞的分泌腔中。在这里，它们准备在随后的血粉中传播给脊椎动物宿主。由于成虫腺体形成为幼虫SG导管芽残留物的形成，这些残余物持续存在于早期蛹SG组织分析之后，因此幼虫SG是限制疾病传播的干预措施的理想靶标。了解幼虫SG的发育有助于更好地了解其形态和功能适应性，并有助于评估针对该器官的新干预措施。该视频实验方案展示了一种有效的技术，用于从 冈比亚按蚊中 分离，固定和染色幼虫SG。在25%乙醇溶液中从幼虫中解剖的腺体固定在甲醇 – 冰醋酸混合物中，然后进行冷丙酮洗涤。在磷酸盐缓冲盐水（PBS）中冲洗几次后，可以用广泛的标记染料和/或抗血清对SG表达的蛋白质进行SG染色。这种用于幼虫SG分离的方法也可用于收集组织，用于 原位 杂交分析，其他转录组学应用和蛋白质组学研究。
Introduction
疟疾是一项重大公共卫生威胁，2019年造成近2.3亿人感染，估计有40.9万人死亡1。大多数死亡发生在撒哈拉以南非洲，是由寄生虫恶性疟原虫引起的，其昆虫载体是冈比亚按蚊，这是本视频演示的主题。尽管这些数字表明，自世纪之交以来，年死亡率大幅下降（年死亡人数减少了30万>），但从2000年到2015年观察到的疾病发病率的有希望的下降正在逐渐减少，这表明需要采取新的方法来限制疾病传播2。控制和可能消除疟疾的有希望的额外策略之一是使用基于CRISPR / Cas9的基因编辑和基因驱动3，4，5来靶向蚊子媒介的能力。事实上，以蚊媒为目标（通过扩大使用经杀虫剂处理的长效蚊帐）对减少疾病传播产生了最大的影响6。
雌性蚊子在血粉期间从受感染的人类那里获得疟原虫配子体。在受精，成熟，中肠上皮穿越，种群扩张和专性蚊子宿主的血细胞导航之后，数百至数万个疟原虫孢子体侵入蚊子SG并填充组成分泌细胞的分泌腔。一旦进入分泌腔，寄生虫就可以直接进入唾液管，因此准备在下一次血粉时传播给新的脊椎动物宿主。由于SGs对于致疟疾孢子体传播给人类宿主至关重要，并且实验室研究表明SGs对于血液喂养，蚊子存活或繁殖力不是必需的7，8，9，它们代表了阻断传播措施的理想目标。成虫SGs是幼虫SGs中”导管芽”残留物的形成，持续超过早期蛹SG组织分析10，使幼虫SG成为限制成虫期疾病传播的理想目标。
表征SG发育的幼虫阶段不仅有助于更好地了解其形态和功能适应性，还可以帮助评估通过关键SG调节因子的基因编辑靶向该器官的新干预措施。由于先前所有关于幼虫唾液腺结构的研究都早于免疫染色和现代成像技术10，11，我们已经开发出一种用各种抗体和细胞标志物分离和染色唾液腺的方案12。本视频演示了从冈比亚按蚊L4幼虫中提取，固定和染色幼虫SGs以进行共聚焦成像的方法。
Protocol
Representative Results
Discussion
本文中描述的方案改编自果蝇SG解剖方案和成年蚊子解剖方案14、15、16。然而，当使用成人解剖和SG染色方法时，大多数标志物没有穿透基底膜（数据未显示）。成人方案的适应性包括在25%EtOH溶液中解剖腺体，用MeOH和冰醋酸的组合洗涤腺体，以及进行90s丙酮洗涤。在最初的成人方案中，在1xPBS14，15，16</sup…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢约翰霍普金斯疟疾研究所获取和饲养 冈比亚 锥虫幼虫。
Materials
| KH2PO4 | Millipore Sigma | P9791 | |
| Na2HPO4 • 2H2O | Millipore Sigma | 71643 | |
| NaCl | Millipore Sigma | S7653 | |
| Acetone | Millipore Sigma | 179124 | |
| Brush with soft bristles | Amazon (SN NJDF) Detail Paint Brush Set | B08LH63D89 | |
| Cover slips (22 x 50 mm) | VWR | 48393-195 | |
| DAPI (DNA) | ThermoFisher Scientific | D1306 | |
| Ethyl alcohol 200 proof | Millipore Sigma | EX0276 | |
| Gilson Pipetman P200 Pipette | Gilson | P200 | |
| Glacial Acetic Acid | Sigma Aldrich | 695092 | |
| Jewelers forceps, Dumont No. 5 | Millipore Sigma | F6521 | |
| KCl | Millipore Sigma | 58221 | |
| Methanol | Millipore Sigma | 1414209 | |
| Nail polish | Amazon (Sally Hansen) | B08148YH9M | |
| Nile Red (lipid) | ThermoFisher Scientific | N1142 | |
| Paper towels/wipes | ULINE | S-7128 | |
| Petri plate (to make putty plate) | ThermoFisher Scientific | FB0875712 | |
| Pipette Tips | Gilson | Tips E200ST | |
| Plastic Transfer Pipette | Fisher Scientific | S304671 | |
| Primary antibodies (e.g., Crb, Rab11) | Developmental Studies Hybridoma Bank (DSHB); Andrew Lab | Mouse anti-Crb (Cq4) or Rabbit anti-Rab11 | |
| Secondary antibodies with fluorescent tags (e.g., Alexa Fluor 488 Goat-anti Rabbit) | ThermoFisher Scientific | A11008 | |
| Silicone resin and curing agent for putty plate | Dow Chemicals – Ximeter Silicone | PMX-200 | |
| Slides, frosted on one end for labelling | VWR 20 X 50 mm | 48393-195 | |
| Wheat Germ Agglutinin | ThermoFisher Scientific | W834 |
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