Jacot J L, Glover J P, Robison W G
Section on Pathophysiology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2740, USA.
Biotech Histochem. 1995 Nov;70(6):277-84. doi: 10.3109/10520299509108333.
The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryoprotective agents, mechanical removal of Descemet's membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at -70 C in O.C.T. compound. The Descemet's membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO4-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO4-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO4-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O.C.T. compound, removal of the Descemet's membrane-endothelium complex, and treatment with alpha amylase reduced non-specific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohistochemical procedures where the reaction products would be obscured by background staining.
本研究的目的是改进用于角膜全层标本中总神经支配研究的氯化金程序，以减少非特异性背景染色，并消除标准氯化金技术中逐渐恶化的染色质量。改进措施包括使用冷冻保护剂、在固定前机械去除后弹力层 - 内皮细胞复合体、用α淀粉酶处理，以及使用含硬化剂的柯达快速定影液停止将氯化金还原为金属金。大鼠角膜在O.C.T. 复合物中于 -70°C保存。解冻后去除后弹力层 - 内皮细胞复合体，角膜用含8%蔗糖的4%磷酸钠缓冲多聚甲醛固定。固定后的角膜在含α淀粉酶的磷酸钠缓冲盐水中孵育，置于100%柠檬汁中，然后置于1%氯化金溶液中，转移至冰醋酸中，再置于快速定影液中，用磷酸钠缓冲盐水冲洗，并用梯度酒精脱水。使用对侧角膜作为对照检查全层角膜的平铺标本。与对照相比，在O.C.T. 复合物中冷冻角膜、去除后弹力层 - 内皮细胞复合体以及用α淀粉酶处理可减少非特异性背景染色。用柯达快速定影液处理可使染色质量至少在8个月内不恶化。这些改进使得氯化金技术能够与免疫组织化学程序一起使用，在这些程序中，反应产物会被背景染色所掩盖。
