DEMONSTRATION OF CHOLINESTERASES BY FORMATION OF OSMIUM BLACKS AT SITES OF HATCHETTS BROWN
DEMONSTRATION OF CHOLINESTERASES BY FORMATION OF OSMIUM BLACKS AT SITES OF HATCHETTS BROWN
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DOI：
10.1007/bf00304184
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发表时间：
1973-01-01
期刊：
影响因子：
--
通讯作者：
MOORE, HG
中科院分区：
文献类型：
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作者：
HANKER, JS;THORNBURG, LP;MOORE, HG
根据 Karnovsky 和 Roots (1964) 的方法，少量 Hatchett 棕色（亚铁氰化铜，Cu2Fe(CN)6·7H2O）沉积在组织中胆碱酯酶活性位点，可通过硫代碳酰肼 (TCH) 与锇桥联来增强。或者，可以通过利用亚铁氰化铜作为催化剂来实现3,3'-二氨基联苯胺(DAB)的氧化偶联，从而实现沉积物的放大。所得的浓色亲锇聚合物更加明显，并且在锇化后，是电子不透明的。通过这些程序，光学显微镜制备比 Koelle 和 Friedenwald 程序的 Gomori 修改更持久。这种方法的主要优点之一是可以拍摄厚的连续塑料切片 (1-2 μm)，并通过光学显微镜轻松研究，直到出现所需的酶染色区域。从而方便了超薄切片区域的选择；通常难以定位的酶染色结构，例如上皮内神经末梢，很容易被发现。强化程序允许在较低温度下缩短孵育时间。这导致原始无定形凝胶状 Hatchett 棕色沉积物聚结成相对较大的立方晶体的趋势减弱。在 Hatchett 棕色形成后，立即用 0.1 M、pH 7.2、三（羟甲基）氨基甲烷 (TRIS) 缓冲液冲洗组织，然后用 TCH 或 DAB 强化，发现在较长的孵育后可有效消除一些较小的背景沉积物。遗传性感觉神经病表现出乙酰胆碱酯酶活性严重耗竭，同时伴有硬腭皱襞感觉末梢的丧失，证实了神经病的外周性质。发现与传入三叉神经成分相关的高水平乙酰胆碱酯酶，特别是含有透明囊泡的感觉末梢，表明它们可能具有胆碱感受性。
Small amounts of Hatchett's brown (cupric ferrocyanide, Cu2Fe(CN)6·7H2O), deposited at the sites of cholinesterase activity in tissues by the procedure of Karnovsky and Roots (1964), may be enhanced by bridging to osmium through thiocarbohydrazide (TCH). Alternatively, amplification of the deposits may be attained by utilizing the cupric ferrocyanide as a catalyst to effect the oxidative coupling of 3,3′-diaminobenzidine (DAB). The resulting intensely colored osmiophilic polymer is more visible and after osmication, is electron opaque. With these procedures, the light microscope preparations are more permanent than with the Gomori modification of the Koelle and Friedenwald procedure.One of the major advantages of this method is that thick serial plastic sections (1–2 μm) may be taken and readily studied by light microscopy until the required enzymatically stained areas appear. Thus the selection of areas for ultrathin sectioning is facilitated; enzymatically stained structures usually difficult to locate, such as intraepithelial nerve endings, are readily found.The intensification procedures permit shorter incubation times at lower temperatures. This results in diminished tendency of the original amorphous, gel-like Hatchett's brown deposits to coalesce into relatively large cubic crystals.Immediate rinsing of the tissues after the formation of Hatchett's brown with a 0.1 M, pH 7.2, tris (hydroxymethyl)aminomethane (TRIS) buffer prior to intensification with either TCH or DAB was found effective in eliminating some of the smaller background deposits after the longer incubations.Studies in developing mice with an hereditary sensory neuropathy showed severe depletion of acetylcholinesterase activity concomitant with the loss of sensory endings from the rugae of hard palate, confirming the peripheral nature of the neuropathy.High levels of acetylcholinesterase found associated with afferent trigeminal components, especially sensory endings containing clear vesicles, suggest that they may be cholinoceptive.