Vectashield antifading solution

Adult liver flukes were flat-fixed in 4% paraformaldehyde and incubated with fluorescein-labeled lectins or with biotin-MAL II followed by incubation with fluorescein-labeled anti-biotin (Vector Laboratories). Parasites were mounted on glass microscope slides with Vectashield® anti-fading solution with or without DAPI (Vector Laboratories). Specimens were viewed using a Leica DM IL LED microscope using 10×, 20×, and 40× HI PLAN I objectives (Leica Microsystems, Ashbourne, Ireland) equipped with epifluorescent source and a filter system for FITC and TRITC fluorescence. Images were processed with Adobe Photoshop CS4 software (Adobe System Inc. San Jose, CA).

The following protocol was adapted from a previously described method [41 ]. Briefly, adult liver flukes were flat-fixed in 4% paraformaldehyde and incubated with fluorescein-labelled ConA (Vector Laboratories). Parasites were mounted on glass microscope slides with Vectashield anti-fading solution (Vector Laboratories). Specimens were viewed using a Leica DM IL LED microscope using 10x, 20x, and 40x HI PLAN I objectives (Leica Microsystems, Wetzlar, Germany) equipped with epifluoresce source and a filter system for FITC fluorescence. Images were processed with Adobe Photoshop CS4 software (Adobe System Inc., San Jose, USA).

The oligo library was synthesized by MYcroarray (Ann Arbor, MI, USA). Probe preparation from the synthesized oligo library was conducted as described by Han et al. [30 (link)]. FISH analysis was performed as described by Han et al. [30 (link)]. The biotin- or digoxigenin-labeled single-stranded oligos prepared from the library were directly used as FISH probes. CentO which contains a 155-bp satellite repeat of a rice centromere [50 (link)], a pTa794 clone containing the coding sequence for the 5S rDNA of wheat [59 (link)], 45S rDNA [47 ] and a pAtT4 clone containing A. thaliana telomeric DNA [60 ] were labeled with either digoxigenin-11-dUTP or biotin-dUTP (Roche) by standard nick translation and included in FISH probes [61 (link)].
Each probe was detected using a fluorescein isothiocyanate conjugated anti-biotin or anti-digoxigenin antibody (Vector Laboratories). The chromosomes were counterstained with 4′, 6′-diamidino-phenylindole (DAPI) in Vectashield antifading solution (Vector laboratories). Chromosomes and FISH signals were observed under an Olympus BX61 fluorescence microscope and images were captured with a DVC1412 CCD camera using IPLab software.