Immunogold-Silver Staining (IGSS) of Agarose Embedded (NCP) Bvdv-Infected Cell Suspensions

Abstract

The use of pre-embedding immunogold-silver staining (IGSS) in electron microscopy of cell culture monolayers has been reported by various authors and its usefulness to localize antigens has been documented. Embedding cell suspensions or cell fractions for IGSS can require time-consuming centrifugation steps. Embedding cell suspensions in agarose simplifies IGSS processing and embedding procedures.Bovine turbinate cells (BTs) were grown to confluence in 25cm3culture flasks. BTs were infected with a non-cytopathic strain (ncp) of bovine viral diarrhea virus (BVDV) while control cells were sham inoculated. At 18 h pi, cells were detached with trypsin. All following procedures were carried out at RT. The cells were fixed for 30 min in 2.0% paraformaldehyde + 0.2% glutaraldehyde in 0.1 M. phosphate buffer, pH 7.4, and rinsed 3 times in 0.1 M phosphate buffer, pH 7.4 (each centrifuged for 10 min at 300g). The cells were resuspended in 1 ml of buffer, transferred to an eppendorf tube and centrifuged for 10 min at 300g. The excess buffer was removed, 1-2 drops of warm, molten 1.0% agarose added, and the mixture centrifuged for 1 min at 300g to pellet the cells to the tip of the tube. The solidified cell-agarose mixture was removed, cut into <0.5mm3pieces, and placed in phosphate buffer.