Department of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
J Neurosci Methods. 2009 Oct 30;184(1):115-8. doi: 10.1016/j.jneumeth.2009.07.010. Epub 2009 Jul 15.
Nissl staining is a widely used method to study morphology and pathology of neural tissue. After standard immunocytochemistry, the Nissl staining labels only the nucleus of neurons and the characteristic staining of the neuronal perikarya is absent or very weak. We hypothesized that the RNA degradation during the immunocytochemical treatment results in the loss of cytoplasmic staining with Nissl-dyes. To test this hypothesis, we used RNAse-free conditions for all steps of immunostaining. To further prevent the RNA-degradation by RNAse contaminations, the RNAse inhibitor heparin was added to all antibody-containing solutions. The efficiency of Nissl staining after standard and RNAse-free double-labeling immunocytochemistry was compared using antibodies against c-Fos and neuropeptide Y (NPY) on tissues of rats refed after 3 days of fasting. After standard immunocytochemistry, the Nissl-staining labeled the nuclei of neurons and only very faintly the cytoplasm of these cells. The RNAse-free treatment did not alter the distribution of immunoreaction signal, but preserved the staining of neuronal perikarya by the Nissl-dyes. In conclusion, the RNAse-free conditions during immunocytochemistry allow the labeling of neuronal perikarya by Nissl-dyes. The described method facilitates the mapping of immunocytochemical signals and makes possible the light microscopic examination of the innervation of neurons identified by their nuclear protein content.
尼氏染色是一种广泛用于研究神经组织形态和病理学的方法。在进行标准免疫细胞化学染色后，尼氏染色仅标记神经元的核，而神经元胞体的特征染色缺失或非常微弱。我们假设免疫细胞化学处理过程中的 RNA 降解导致尼氏染料对细胞质的染色丢失。为了验证这一假设，我们在免疫染色的所有步骤中都使用了无 RNA 酶的条件。为了进一步防止 RNA 酶污染导致的 RNA 降解，我们在含有抗体的所有溶液中添加了 RNA 酶抑制剂肝素。我们使用针对 c-Fos 和神经肽 Y（NPY）的抗体，对禁食 3 天后重新进食的大鼠组织进行标准和无 RNA 酶双重免疫细胞化学染色，比较了两种方法后的尼氏染色效率。在标准免疫细胞化学染色后，尼氏染色标记了神经元的核，而这些细胞的细胞质仅非常微弱地染色。无 RNA 酶处理并未改变免疫反应信号的分布，但保留了尼氏染料对神经元胞体的染色。总之，免疫细胞化学过程中的无 RNA 酶条件允许尼氏染料标记神经元胞体。该方法有助于免疫细胞化学信号的定位，并使通过核蛋白含量鉴定的神经元的神经支配的光镜检查成为可能。
