A simplified method for the detection of maedi-visna virus RNA by in situ hybridization.
A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.
Roy
D J
DJ
Department of Veterinary Pathology, Edinburgh University, U.K.
Watt
N J
NJ
Ingman
T
T
Houwers
D J
DJ
Sargan
D R
DR
McConnell
I
I
D000096:Acetone; D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D002460:Cell Line; D005557:Formaldehyde; D007150:Immunohistochemistry; D008722:Methods; D009693:Nucleic Acid Hybridization; D011108:Polymers; D012367:RNA, Viral / Q000032:analysis*; D012756:Sheep; D014790:Visna-maedi virus / Q000302:isolation & purification*
D000956:Antigens, Viral; D011108:Polymers; D012367:RNA, Viral; D000096:Acetone; D005557:Formaldehyde; C003043:paraform