Histochemical localization of gene expression in Onchocerca volvulus: in situ DNA histohybridization and immunocytochemistry.
We report here the development of in situ hybridization and immunohistochemistry protocols which permit the histological identification of gene expression of a cloned antigen of Onchocerca volvulus, OI5, in the parasite. Skin nodules containing female adult worms were fixed in a modified Carnoy's fixative and embedded in paraffin. Histological staining of tissue sections revealed uniformly excellent morphology and RNA preservation. To localize mRNA by in situ hybridization, tissue sections were incubated with biotin-labeled pOI5, the plasmid containing the genomic sequence of the antigen, and hybridization signals were histochemically visualized using a streptavidin-enzyme conjugate and chromogenic substrates. The protein antigen was localized immunohistochemically by incubating the sections with specific antibodies prepared against a recombinant fusion protein containing the OI5 sequence (OI3), and visualized via a secondary antibody-biotin-enzyme conjugate procedure. The results reported here showed distinct localization of the OI5 mRNA and OI3 antigen in specific cellular and tissue regions of the adult parasite, and in microfilariae located within the uteri and in the surrounding host tissue. The specificity and high sensitivity of these histological detection methods should be generally applicable for the characterization of gene expression in the filarial parasite, particularly the insect-borne, infective filarial larvae, which are severely limited in quantity.
Tuan
R S
RS
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107.
Shepley
K J
KJ
Mulligan
M M
MM
Abraham
D
D
Perler
F B
FB
D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics; D004247:DNA / Q000378:metabolism; D015342:DNA Probes; D005260:Female; D015870:Gene Expression; D006652:Histological Techniques; D006801:Humans; D009693:Nucleic Acid Hybridization; D009854:Onchocerca / Q000235:genetics*; D012867:Skin / Q000469:parasitology
D000947:Antigens, Helminth; D015342:DNA Probes; D004247:DNA