Immunochemistry was performed on fixed and sectioned B. malayi parasites recovered at various stages of development from intraperitoneally infected jirds, as well as on fixed and sectioned O. volvulus nodules, which were originally obtained from an onchocerciasis patient in Ghana [33 (link),34 (link)]. The parasites were fixed in either 4% formaldehyde or in 80% ethanol then embedded in paraffin. Immunostaining was conducted with the alkaline phosphatase anti-alkaline phosphatase (APAAP) method as previously described [33 (link)]. Primary antibody dilutions of 1:100 to 1:1000 were assessed, and the dilutions 1:200 and 1:500 were found to provide the best signal over background. Polyclonal rabbit anti-mouse IgG 1:25 (Dako, Santa Clara CA, USA) was used as the secondary antibody, then mouse APAAP of at a dilution of 1:40 (Sigma) was applied. The chromatogen Fast Red (Sigma) was used as the substrate and the slides were counter stained with hematoxylin (Merck, Darmstadt Germany). Slides were examined on an Olympus -BX40 microscope and photographed on an Olympus DP70 microscope digital camera (Olympus, Tokyo Japan).
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