Schneider H
Universität Konstanz, Fakultät für Biologie, F.R.G.
J Neurosci Methods. 1989 Nov;30(2):107-15. doi: 10.1016/0165-0270(89)90056-3.
The bright red fluorescent dye, Texas red, is introduced for ionophoretic microinjection in conjunction with the well-known dye Lucifer yellow. Different identified neurones can thus be visualised in two strongly contrasting colours in the same preparation (differential intracellular staining) following their physiological characterisation. Satisfactory results were obtained with electrodes filled with 4% Texas red (sulforhodamine 101 acid chloride; w/v) in 1 M potassium acetate (pH 3.0) and 5% Lucifer yellow (w/v) in aqua dest., respectively. Texas red was injected ionophoretically with pulsed depolarising current (3-10 nA, 500 ms pulses at 1 Hz, 15-30 min) and Lucifer yellow with hyperpolarizing constant current (5-6 nA, 5-15 min). Histological tissue processing was identical for both dyes, the quality of intracellular recordings with Texas red electrodes was similar to that with Lucifer yellow electrodes. Stained neurones could be visualised in both whole-mounts and sectioned preparations. Differential staining of two identified synaptically coupled neurones, a motoneurone and an interneurone, in the mesothorax of Locusta is presented as an illustration for the possible localisation of contact sites at the light-microscopic level.
将亮红色荧光染料德克萨斯红与著名的染料路西法黄一起用于离子电泳显微注射。这样，在对不同的已识别神经元进行生理学特征描述后，就可以在同一标本中用两种强烈对比的颜色将它们可视化（差异细胞内染色）。分别用填充有4%德克萨斯红（磺基罗丹明101酰氯；重量/体积）的1M醋酸钾（pH 3.0）电极和5%路西法黄（重量/体积）的去离子水电极获得了满意的结果。德克萨斯红通过脉冲去极化电流（3 - 10 nA，1 Hz下500 ms脉冲，15 - 30分钟）进行离子电泳注射，路西法黄通过超极化恒流（5 - 6 nA，5 - 15分钟）进行注射。两种染料的组织学处理相同，使用德克萨斯红电极进行细胞内记录的质量与使用路西法黄电极的相似。染色的神经元在整装标本和切片标本中都可以可视化。作为在光学显微镜水平上可能定位接触位点的一个例子，展示了对蝗虫中胸的一个运动神经元和一个中间神经元这两个已识别的突触耦合神经元的差异染色。
