Immunohistochemical Staining Methods for Connexin32 on Formalin-Fixed Paraffin-Embedded Sections

Connexin32 (Cx32), one of the components of gap junction intercellular communication, is commonly detected by the fluorescent antibody method, although this method has some defects when conducting detailed histopathological investigations. In order to develop a new method to improve the fluorescent antibody method, the optimal immunohistochemical procedure on formalin-fixed paraffin-embedded sections was examined. According to the results, the localization of Cx32 was observed on sections prepared with the Dako catalyzed signal amplification (CSA) method after 0.2% protease digestion for 30 minutes after a 3-day or 6-month fixation. As the CSA method is highly sensitive and based on signal amplification by biotinylated tyramide, the localization of Cx32 was clearly shown on the cell membranes of hepatocytes. In fact, by this CSA method, a decrease in the signals of Cx32 was shown in the liver of rats treated with phenobarbital or in hepatocellular adenoma, which were consistent with previous reports using cryosections. In conclusion, the CSA method is considered an appropriate method for the detection of Cx32 in formalin-fixed paraffin-embedded sections.