Traditionally tissues for in situ hybridization of viral nucleic acid have been small pieces obtained from laboratory rodents, and fixatives that are designed for electron microscopy, such as periodate-lysine-paraformaldehyde (PLP) can handle them adequately. However, these fixatives have limited penetrating ability and may produce no appreciable hardening, so alternative fixation methods were evaluated. The intention was to determine whether fixatives adequate for bulky tissues such as whole or halved pig and cow brains would also be compatible with in situ hybridization. Various fixatives were evaluated using a system of intracranial inoculation of BALB/c mice with pseudorabies virus (PRV) followed by in situ hybridization of brain tissue sections with a 35S-labeled PRV DNA probe. Loss of tissue sections was a major problem, particularly with PLP and formalin, but positive results were obtained with five fixatives tested. Cellular morphology was especially good with PLP and with a modification of Carnoy's fluid, MOCA fixative. An incidental but important observation was that formalin is compatible with in situ hybridization. Retroactive studies of viral diseases using routinely processed blocks of tissue (formalin-fixed, paraffin-embedded) are therefore conceivable.
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