Tzaneva Velislava, Perry Steve F
Department of Biology, University of Ottawa;
Department of Biology, University of Ottawa.
J Vis Exp. 2015 Mar 19(97):52548. doi: 10.3791/52548.
Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the gill where they transport ions such as Na+, Cl- and Ca2+ via a variety of ion channels, pumps and exchangers. The teleost gill is extrinsically innervated by the facial (VI), glossopharyngeal (IX) and vagus (X) nerves. The IX and X nerves are also the extrinsic source of branchial IC innervation. Here, two techniques used to study the innervation, proliferation and distribution of ICs are described: a time differential staining technique and a full bilateral gill denervation technique. Briefly, goldfish are exposed to a vital mitochondrion-specific dye (e.g., MitoTracker Red) which labels (red fluorescence) pre-existing ICs. Fish were either allowed to recover for 3-5 days or immediately underwent a full bilateral gill denervation. After 3-5 days of recovery, the gills are harvested and fixed for immunohistochemistry. The tissue is then stained with an α-5 primary antibody (targets Na+/K+ ATPase containing cells) in conjunction with a secondary antibody that labels all (both new and pre-existing) ICs green. Using confocal imaging, it was demonstrated that pre-existing ICs appear yellow (labelled with both a viable mitochondrion-specific dye and α-5) and new ICs appear green (labelled with α-5 only). Both techniques used in tandem can be applied to study the innervation, proliferation and distribution of ICs on the gill filament when fish are exposed to environmental challenges.
鳃离子细胞（ICs）是鱼类离子调节的功能单位。在成鱼中，它们存在于鳃的丝状和片状上皮上，通过多种离子通道、泵和交换器运输Na+、Cl-和Ca2+等离子。硬骨鱼的鳃由面神经（VI）、舌咽神经（IX）和迷走神经（X）进行外部神经支配。IX和X神经也是鳃IC神经支配的外部来源。在此，描述了两种用于研究ICs神经支配、增殖和分布的技术：时间差异染色技术和全双侧鳃去神经支配技术。简要地说，将金鱼暴露于一种重要的线粒体特异性染料（例如，MitoTracker Red），该染料标记（红色荧光）预先存在的ICs。让鱼恢复3 - 5天或立即进行全双侧鳃去神经支配。恢复3 - 5天后，收获鳃并固定用于免疫组织化学。然后用α - 5一抗（靶向含Na+/K+ ATP酶的细胞）结合标记所有（新的和预先存在的）ICs为绿色的二抗对组织进行染色。使用共聚焦成像表明，预先存在的ICs呈现黄色（用活性线粒体特异性染料和α - 5标记），新的ICs呈现绿色（仅用α - 5标记）。当鱼暴露于环境挑战时，串联使用的这两种技术都可用于研究鳃丝上ICs的神经支配、增殖和分布。
