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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.  相似文献
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采用扫描电镜观察了不同碱度(0、2、4 g/L NaHCO3)胁迫对尼罗罗非鱼(Oreochromis niloticus)鳃离子细胞形态变化的影响,并采用免疫组化技术观察了鳃、肾、肠中4个HCO3-转运因子碳酸酐酶(CA II、CAⅣ)、碳酸氢钠协同转运载体(SLC4A4)、Cl-/HCO3-离子交换体(SLC26A6)的阳性反应变化.扫描电镜结果表明,鳃离子细胞分布在鳃小片基部.根据其表面开孔形状和尺寸,可分为Ⅰ型、Ⅱ型、Ⅲ型和Ⅳ型4种亚型,各亚型离子细胞的开孔尺寸随碱度胁迫强度增高呈正比增大,Ⅲ型离子细胞开孔尺寸变化最明显(P<0.01);离子细胞总数目也随碱度升高而增加,Ⅲ型离子细胞数目上升最为显著(P<0.01).免疫组化结果表明,在淡水、碱水组中,CA Ⅱ、CAⅣ、SLC4A4、SLC26A6在鳃小片基部和肾中均有阳性反应,且随着碱度升高,阳性反应增强,但在肠道中未观察到阳性反应.本研究结果初步表明,尼罗罗非鱼可通过鳃离子细胞形态和数量调节适应碱度变化,鳃和肾为主要应答调节器官.  相似文献
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