Ultrastrctural identification of viral infections in paraffin sections stained by immunohistochemistry and in situ hybridization

Abstract

Confirmation of viral infections in surgical biopsies is sometimes limited to routine paraffin sections because a specimen may not have been set aside for culture or culture results are negative. Since viral infections are frequently focal and localized to only a few cells, definitive histopathological diagnosis can be difficult to achieve even with immunohistoGhemistry (IHC), in situ hybridization (ISH) or electron microscopy. In this paper, we would like to demonstrate a siirple and direct approach to confirming viral infections in paraffin sections , using two immuno-labelling techniques coupled with electron microscopy.Paraffin sections of surgical specimens are deparaffinized in xylene and rehydrated through graded ethyl alcohol. Herpes simplex virus (Figure 1) and Toxoplasma gondii are localized immunohistochemically by the peroxidase antiperoxidase technique and Pneumocystis carinii (Figure 2) by 3 step ipdirect method, using 3,3'-diaminobenzidine tetrahydrochloride as the chrmogen, CMV ENA probe (ENZO Biochemicals) is used for in situ hybridization, following kit instructions (Figure 3 & 4). After the final chromogen reaction, wet sections are examined with a light microscope. Slides in vrfiich focal positive staining is difficult to interpret as specific are fixed in osmium tetroxide, dehydrated with graded acetone, and infiltrated with Poly/Bed 812. After polymerization, the epoxy blocks containing area of interest can be easily separated from the glass slides by heating on a 100 C hotplate for approximately 20 seconds . The area of interest should be positioned in the center of the block face for ultrathin sectioning. With careful adjustments of knife and microtcme, the area of interest can be precisely sectioned without loss of tissue. Ultrathin sections are collected on uncoated mesh grids for higher contrast and then directly examined by TEM. Peroxidase-labelled microorganisms or viral DHA can be easily localized and identified without heavy metal staining. Cellular details can be further increased by staining with uranyl acetate and lead citrate. Tissue remaining in block face after sectioning is available for IM comparison with the EM micrographs.