Department of Anatomy, Faculty of Medicine, University of Szeged, Szeged, Hungary.
Department of Medical Chemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary.
J Histochem Cytochem. 2020 Aug;68(8):571-582. doi: 10.1369/0022155420942213. Epub 2020 Jul 13.
Double staining protocols using the most popular immunoperoxidase techniques may raise difficulties. The two ordinary detection systems may cross-talk, when the primary antibodies are derived from phylogenetically closely related animals. A color shift of the 3,3'-diaminobenzidine (DAB) polymer may occur during the second development, resulting in poor distinction between the two kinds of deposits. A post-DAB technique, sulfide-silver-gold intensification, was fine tuned to eliminate these difficulties, which may be especially suitable for colocalization of cell nuclei and perikarya of the same cells. The revised method was probed in combination with a subsequent other immunoperoxidase step or fluorochrome-tagged reagents. The nuclear antigens (BrdU, c-Fos, and Prox-1) were first visualized with DAB polymer, which were then treated with SSGI, turning the deposit black. Thereafter, cytoplasmic antigens (doublecortin, neuronal nuclei, and calbindin) were detected with either another immunoperoxidase using DAB again or immunofluorescence labeling. In both approaches, the immunopositive nuclei and cytoplasmic sites could be easily distinguished even at low magnifications. Different shielding or eluting posttreatments were compared for consecutive acetylcholinesterase histochemistry terminated with DAB development and immunohistochemistry in the same sections. In conclusion, we recommend post-DAB treatments that abolish interactions between detection systems and allow clear distinction between the two signals under various conditions.
使用最流行的免疫过氧化物酶技术的双重染色方案可能会带来困难。当两种主要抗体来源于系统发育上密切相关的动物时，两种普通的检测系统可能会发生串扰。在第二次显色过程中，3,3'-二氨基联苯胺（DAB）聚合物的颜色可能会发生偏移，从而导致两种沉积物之间的区分不明显。过 DAB 技术，硫化银金增强，被微调以消除这些困难，这可能特别适合于同一细胞的细胞核和胞体的共定位。该改良方法与随后的另一种免疫过氧化物酶步骤或荧光标记试剂联合进行了探测。首先使用 DAB 聚合物可视化核抗原（BrdU、c-Fos 和 Prox-1），然后用 SSGI 处理，使沉积物变黑。此后，用 DAB 再次进行另一种免疫过氧化物酶检测，或免疫荧光标记，检测细胞质抗原（双皮质素、神经元核和钙结合蛋白）。在这两种方法中，即使在低倍放大下，也可以轻松区分免疫阳性核和细胞质部位。比较了不同的屏蔽或洗脱后处理方法，用于在同一切片中用 DAB 显色终止的连续乙酰胆碱酯酶组织化学和免疫组织化学。总之，我们建议使用过 DAB 处理，以消除检测系统之间的相互作用，并在各种条件下清晰地区分两种信号。
