Medical Laboratory Science Program, Department of Health Sciences, Illinois State University, Normal, IL, USA.
Department of Psychology and School of Biological Sciences, Illinois State University, Normal, IL, USA.
J Anat. 2019 Jun;234(6):936-942. doi: 10.1111/joa.12967. Epub 2019 Mar 12.
c-Fos is the product of a gene expressed within neurons in the brain that serves as an anatomical marker of cellular activation. Immunohistochemical staining for c-fos allows a characterization of the effects of many different types of experimental manipulations on neuronal activity, making it a powerful technique for understanding brain, drug and behavior relationships. This study compared visualization of an anti-c-fos primary antibody in 40-μm-thick cryostat sections of formaldehyde-fixed rat brainstem using either a peroxidase enzyme-conjugated secondary antibody (indirect peroxidase) or the peroxidase-conjugated avidin-biotin complex (ABC) method. All sections were treated with H O to quench endogenous peroxidase enzyme and sodium borohydride to enhance permeability of the tissue and improve staining quality. Every other section was used to examine either the indirect peroxidase or the ABC method. Sections for the indirect peroxidase method were treated with Triton X-100 detergent to increase tissue permeability, goat serum to reduce non-specific binding of the secondary antibody and, in some cases, bovine serum albumin (BSA) to reduce non-specific binding of the primary antibody. Sections for the ABC method were treated with dilute normal serum, and avidin and biotin solutions and, in some cases BSA. Alternate sections were incubated for 72 h in either rabbit anti-c-fos primary antibody (1 : 20 000) or its vehicle (negative control). For the indirect peroxidase protocol, tissues were treated with peroxidase-conjugated goat anti-rabbit secondary antibody. For the ABC protocol, tissues were treated with biotinylated goat anti-rabbit secondary antibody and ABC peroxidase complex. All sections were reacted with 3,3'-diaminobenzadine (DAB) and H O , mounted and coverslipped. Both methods produced specific staining of c-fos-containing neurons, relative to the negative control sections. The indirect peroxidase protocol produced clear staining of c-fos-containing neurons, with very little background in the negative control sections. Staining for c-fos was enhanced using the ABC method in that c-fos stained neurons were darker and more clearly visible after shorter treatment with DAB. However, negative control sections showed a greater amount of non-specific staining with the ABC method. Thus, the ABC method was more sensitive but showed reduced specificity, with BSA treatment slightly reducing the level of non-specific staining. Overall, the ABC method produced better visualization and contrast of c-fos-containing neurons against the background color of the tissue.
c-Fos 是一种在大脑神经元中表达的基因产物，作为细胞激活的解剖学标记。c-fos 的免疫组织化学染色允许对许多不同类型的实验操作对神经元活动的影响进行特征描述，使其成为了解大脑、药物和行为关系的强大技术。本研究比较了在福尔马林固定的大鼠脑干 40μm 厚的冰冻切片中使用过氧化物酶酶结合的二级抗体（间接过氧化物酶）或过氧化物酶结合的亲和素-生物素复合物（ABC）方法对抗 c-fos 一抗的可视化。所有切片均用 H2O2 处理以淬灭内源性过氧化物酶酶，并用硼氢化钠处理以增强组织的通透性并提高染色质量。每隔一个切片用于检查间接过氧化物酶或 ABC 方法。用于间接过氧化物酶方法的切片用 Triton X-100 去污剂处理以增加组织通透性，用山羊血清处理以减少二级抗体的非特异性结合，在某些情况下用牛血清白蛋白（BSA）处理以减少一抗的非特异性结合。用于 ABC 方法的切片用稀释的正常血清处理，并用亲和素和生物素溶液处理，在某些情况下用 BSA 处理。交替切片在兔抗 c-fos 一抗（1:20000）或其载体（阴性对照）中孵育 72h。对于间接过氧化物酶方案，组织用过氧化物酶结合的山羊抗兔二级抗体处理。对于 ABC 方案，组织用生物素化山羊抗兔二级抗体和 ABC 过氧化物酶复合物处理。所有切片均用 3,3'-二氨基联苯胺（DAB）和 H2O2 反应，然后安装并盖上盖玻片。与阴性对照切片相比，两种方法均产生了含有 c-fos 的神经元的特异性染色。间接过氧化物酶方案产生了含有 c-fos 的神经元的清晰染色，而阴性对照切片中的背景很少。使用 ABC 方法可以增强 c-fos 的染色，因为在用 DAB 处理较短时间后，c-fos 染色的神经元变暗且更清晰可见。然而，ABC 方法的阴性对照切片显示出更多的非特异性染色。因此，ABC 方法更敏感，但特异性降低，BSA 处理略微降低了非特异性染色的水平。总的来说，ABC 方法产生了更好的可视化效果和对比度，使含有 c-fos 的神经元与组织的背景颜色形成对比。
