Equine herpesvirus 1 causes equine rhinopneumonitis. In order to establish the in situ hybridization (ISH) technique for the detection of equine herpesvirus 1-glycoprotein B (EHV1-gB) gene-derived nucleic acid, we compared the ISH staining intensity in EHV1-infected equine fetal liver tissues fixed with four different fixatives. After being digested with proteinase K (PK), the tissue sections were covered with digoxigenin (DIG)-labeled double stranded DNA probe which hybridize with EHV1-gB gene-derived nucleic acid. After stringent washing, the sections were reacted with alkali-phosphatase-conjugated anti-DIG antibody and then with the enzyme substrate, and examined under microscope. When the tissues were fixed with 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA), an intense positive reaction for EHV1 was seen in the liver cells in the tissue sections predigested with PK. Positive staining in the nuclei and cytoplasm of liver cells were reduced by predigestion of the tissue sections with DNase and RNase, respectively. Bouin-fixation for 2 hours generated a definite positive staining, but the positive staining was minimized in the tissues fixed for 1 day or more. Methacarn-fixation led to a constant positive staining irrespective of fixation time and did not require PK digestion of tissue sections. Ten percent NBF and 4% PFA fixation produced a stronger positive staining than Bouin- and Methacarn-fixation. This study demonstrated that formaldehyde-fixation of tissue samples is the most appropriate for ISH of the EHV1-gB gene. The established ISH technique may be useful for examining the tissue tropism and pathogenesis of EHV1. (Infectious Disease Pathology Section, Department of Infectious Diseases TEL +81-29-838-7837)
