毫微米胶体金、银加强法和锇酸固定在神经组织包埋前免疫电镜术中的应用

Use of Nanogold Followed by Silver Enhancement for Preembedding Immunochemistry in Osmium-fixed Tissue

对于神经递质和受体的精确定位需要有可靠、敏感和高分辨率的包埋前免疫电镜技术。近年来,包埋前毫微米胶体金(直径1.4毫微米)和银加强法开始应用于神经科学研究。然而,存在着金-银颗粒在锇酸处理后明显减小的问题。我们发现包埋前免疫细胞化学染色中经过10分钟银加强的金-银颗粒数目,在锇酸处理后明显地减少。但是,经过2次或3次10分钟银加强的组织中,金-银颗粒的数目和大小都没有明显变化,神经递质和受体可以被金-银颗粒精确定位。这些结果提示大于300平方毫微米的银加强后的金颗粒在锇酸中稳定,可应用于免疫反应阳性物质的精确定位。

A reliable, sensitive, high resolution preembedding immunoelectron microscopy is required to visualize the precise location of neurotransmitters and their receptors. Recently, use of the nanogold (1.4 nm in diameter)followed by silver enhancement for preembedding electron microscopy has been introduced. However, it has been found that the number of the gold grains decreased significantly, when 10 min-intensified nanogolds are treated whir osmium fixation. We check the silver-intensified gold on formvar-coated grids by electron microscopy, and analyze the number and the sizes of the gold grains. The size of the nanogold intensified by silver varies. Eighty five percent of the gold grains intensified with silver for 10 min have sizes smaller than 100 nm^2 and only 41% of them remain after OsO_4 treatment. About seventy percent of the gold grains intensified with silver for 10 min×2 of 10 min×3 have sizes larger than 100 nm^2 and smaller than 300 nm^2. Over 90% of them stand the OSO_4 treatment and largely maintain the same sizes. We have also tested this procedure in a preembedding immunocy-tochemical staining in the sections of the brain and the spinal cord. Both the number and the size of the gold grains intensified with silver for 10 rain are markedly reduced after OsO_4 treatment and empty washed away spots are often seen under electron microscope. Sometimes the immunostaining completely fade away within 5 min in 1% OsO_4. However, the number and the size of the gold grains intensified with silver for 10 min×2 remain basically unchanged after OsO_4 treatment. The location of neuropeptides and receptors can thus be identified. These results suggest that the size of the gold-silver grains above 300 nm^2 are stable in OsO_4 and can be used for localization of immunoreactivity.