Pre-embedding immunogold-silver (IGSS) techniques are useful to localize antigens in cell monolayers and agarose embedded cell suspensions for transmission electron microscopy. Procedural centrifugations, however, present a challenge when attempting to localize antigens in subcellular fractions. Using a Beckman Airfuge Ultracentrifuge to concentrate the subcellar fraction bands and resuspending the organelles in agarose simplifies IGSS processing and resin embedding procedures.Control bovine turbinate (BT), and BT cells infected with cytopathic (cp NADL) and non-cytopathic (ncp NY-1) strains of bovine viral diarrhea virus (BVDV) were fractioned according to Bienz et al (1992). Bands containing membrane vesicles (Fig 2) were collected and each fraction band was pelleted at 169,000g for 20 min using an A-95 fixed angle rotor in a Beckman Airfuge Ultracentrifuge. Each fraction pellet was resuspended in 50μl of 30% agarose, solidified, and trimmed to < lmm. IGSS procedures were carried out according to Nanoprobes, Inc., Stoney Brook, NY, and Hearne & Van Campen (1996).
