Peng Z C, Chen S, Bentivoglio M
Institute of Anatomy and Histology, University of Verona, Italy.
Brain Res Bull. 1995;36(1):101-5. doi: 10.1016/0361-9230(94)00125-k.
Immunostaining of Fos, the nuclear protein encoded by the immediate early gene c-fos, is widely used to reveal the functional activation of neurons. The chemical identity of cells that express c-fos can be investigated with double immunohistochemistry. We report the usefulness of a sequential two-color avidin-biotin-immunoperoxidase method that provides a highly sensitive double immunostaining and allows long-term storage of the sections. In this protocol, metal intensification of diaminobenzidine (int-DAB) resulted in dark brown/black Fos immunostaining of the neuronal nucleus. The use of alpha-naphthol/pyronin reaction product yielded pink immunostaining of a second antigen in the cytoplasm. This combination produced higher contrast than that produced by int-DAB Fos immunostaining combined with conventional DAB light brown cytoplasmic staining. The sensitivity of the use of int-DAB and alpha-naphthol/pyronin was verified in different experimental paradigms, combining the immunocytochemical detection of Fos with that of the p75 nerve growth factor receptor, or parvalbumin, or calbindin D28k.
即刻早期基因c-fos编码的核蛋白Fos的免疫染色被广泛用于揭示神经元的功能激活。表达c-fos的细胞的化学特性可以通过双重免疫组织化学来研究。我们报告了一种顺序双色抗生物素蛋白-生物素-免疫过氧化物酶方法的实用性，该方法可提供高度敏感的双重免疫染色，并允许长期保存切片。在此方案中，二氨基联苯胺的金属强化（int-DAB）导致神经元细胞核的Fos免疫染色呈深棕色/黑色。使用α-萘酚/派洛宁反应产物可在细胞质中产生第二种抗原的粉红色免疫染色。这种组合产生的对比度高于int-DAB Fos免疫染色与传统DAB浅棕色细胞质染色组合产生的对比度。在不同的实验范式中验证了使用int-DAB和α-萘酚/派洛宁的敏感性，将Fos的免疫细胞化学检测与p75神经生长因子受体、小白蛋白或钙结合蛋白D28k的检测相结合。
