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Immunogold-Silver Staining (IGSS) of (NCP and CP) BVDV Infected Subcellular Fraction Bands

Published online by Cambridge University Press:  02 July 2020

C.E. Hearne ,
D.L. Johnson  and
H. Van Campen
C.E. Hearne
Affiliation:
Department Veterinary Sciences, University of Wyoming, Laramie, WY82071
D.L. Johnson
Affiliation:
Department Veterinary Sciences, University of Wyoming, Laramie, WY82071
H. Van Campen
Affiliation:
Department Veterinary Sciences, University of Wyoming, Laramie, WY82071
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Extract

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).

Type
Cytochemistry, Histochemistry, Immunocytochemistry, and In Situ Hybridization
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 155 - 156
Copyright
Copyright © Microscopy Society of America 1997

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References

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5.The authors wish to thank Dr.Dubovi, E.J., New York State Diagnostic Laboratory, Cornell University, Ithaca, NY for providing the MAb 20.10.6 used in this investigation.Google Scholar