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蜜蜂组织中的组织学基础和细胞死亡检测
Chapters
Summary July 7th, 2022
Please note that all translations are automatically generated.
Click here for the English version.
免疫组织化学方法可用于蜜蜂研究，以检测和评估成年蜜蜂中肠和下咽腺的细胞凋亡和坏死水平。
Transcript
该方案在用杀虫剂处理的蜜蜂的许多生物学和毒理学研究中很有用。它有助于检测杀螨剂蜜蜂组织的细胞反应，这些杀螨剂用于蜂群中用于治疗瓦螨。它是一种强大的工具，用于检测与蜜蜂或其他有益昆虫的行为相结合的对组织的亚小影响。
首先，小心地用镊子取一只工蜂，将其放在零下 20 摄氏度的冰上或冰箱中两分钟以使其固定。将蜜蜂斜着固定在培养皿上，从左到右和从右到左两次穿过胸部的最上背部。可怜的昆虫盐水覆盖身体。
将培养皿放在体视显微镜下，聚焦并调整。要解剖中肠，请从腹部开始，将剪刀的一个点插入蜜蜂身体右侧中心的 tergite A5 下方。切到特吉特 A2。保持剪刀内刃与身体侧面平行，以免损坏内脏。
将剪刀向左转并剪。轻轻打开腹部的左侧并固定。用一只手用镊子轻轻向上拉蜜蜂胃，另一只手用剪刀在食道的最末端剪开。
将胃和中肠从腹部拉开，并在直肠处切开。使用带有昆虫盐水溶液的移液管，并去除任何粪便或组织部分。为了解剖下咽腺，如前所述将工蜂固定在冰上。
切下头部并将其放在较小的板上，天线朝上。用两个针固定头部，一个穿过左复眼，第二个穿过右复眼。在针脚内侧的第一个复眼上切开，继续切到盂唇，然后在另一侧穿过第二个复眼进行另一切口。
切断触角。取下面膜，切下仍附着的地方。拿起镊子，小心地去除腺体以及大脑和部分复眼。
对于苏木精和伊红染色，将脱蜡、再水化的切片放入苏木精中五分钟。然后，小心地将它们放在流动的自来水中两分钟。然后，将它们放入蒸馏水中一分钟，将伊红放入四分钟。
将载玻片96%乙醇放置一分钟，然后放入2-丙醇两分钟，最后放入清除剂中两分钟。加入安装介质和盖玻片，让它们干燥。在光学显微镜下观察。
准备科普林罐。准备蛋白酶-K。对切片进行脱蜡和再水化后，将载玻片放入PBS中五分钟。
将载玻片平放在容器中并加入蛋白酶-K。用蒸馏水清洗载玻片。在室温下在内源性过氧化物酶中淬灭。
用PBS或水冲洗载玻片。将载玻片平放在容器中，并在室温下应用平衡缓冲液10秒钟。擦拭组织周围后，向每个切片加入末端脱氧核苷酸转移酶，并在37摄氏度的加湿室中孵育一小时。
孵育前，将湿纸巾放在载玻片周围的托盘内，并用保鲜膜覆盖。孵育后，将标本放在架子上，并将其留在停止洗涤缓冲液中。在PBS中清洗载玻片并在组织周围擦拭后，向切片中加入两滴温热的抗地高辛过氧化物酶偶联物，并在加湿的容器中孵育30分钟。
在PBS中洗涤后，准备工作强度-过氧化物酶底物，用过氧化物酶底物覆盖切片，染色5分钟。将载玻片放在显微镜下并确定最佳染色时间。在染色架和蒸馏水中洗涤载玻片后，将载玻片在蒸馏水中孵育五分钟。
使用苏木精复染两分钟。将载玻片放在流动的自来水中三分钟，然后在蒸馏水中清洗载玻片。将载玻片安装在玻璃盖玻片和安装介质下，平放晾干。
在光学显微镜下观察。使用光学显微镜计算受影响细胞的百分比。结果表明，草酸处理显著影响中肠细胞。
免疫染色显示下咽腺凋亡核中呈阳性红色或棕色反应产物。在大多数腺细胞中测定吡虫啉或香豆磷处理后的阳性反应产物。抬起蜂头面罩时，请注意不要损坏或拉下咽腺的部分。
该技术可检测蜜蜂的亚临床变化，也适用于检测对蜜蜂和其他一些生物体的其他环境影响。
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