Optimal enhancement of in situ hybridization for the detection of porcine circovirus 2 in formalin-fixed, paraffin-wax-embedded tissues using a combined pretreatment of thermocycler and proteinase K.
Optimal enhancement of the hybridization signal was developed for the detection of porcine circovirus (PCV) 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The hybridization signal obtained after thermocycler pretreatment was very uniform across the section, whereas the signal obtained after either proteinase K or microwave pretreatment not only was weaker but was of variable intensity across sections. Thermocycler pretreatment combined with brief proteinase K digestion can enhance signal detection for target viral nucleic acid in formalin-fixed, paraffin-wax-embedded tissues. A strong hybridization signal was detected in the cytoplasm of macrophages and multinucleated giant cells in lymph node and spleen without background staining and morphological damage. The technical improvement results, therefore, in an identical background at the same time as an increased signal and, thus, may help detect lower levels of PCV2 DNA in formalin-fixed, paraffin-wax-embedded tissues.
Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Kwanak-Gu, 151-742 Seoul, Republic of Korea.
J
J
Kim
C
C
Chae
D000818:Animals; D018173:Circoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D017925:Circovirus / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D019286:Endopeptidase K / Q000737:chemistry* / Q000378:metabolism; D017403:In Situ Hybridization / Q000379:methods / Q000662:veterinary*; D016612:Paraffin Embedding / Q000379:methods / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000662:veterinary; D013154:Spleen / Q000821:virology; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology*; D016707:Tissue Fixation / Q000379:methods / Q000662:veterinary; D019282:Wasting Syndrome / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology
D004279:DNA, Viral; D019286:Endopeptidase K