Chromogenic in situ Hybridization for Avian Plasmodium Detection

Chromogenic in situ hybridization ISH was carried out according to Dinhopl et al. [8 (link)]. In brief, 3 μm paraffin wax-embedded tissue sections were subjected to proteolytic treatment with proteinase K (Roche, Basel, Switzerland) 6 μg/ml in Tris-buffered saline at 37 °C for 50 min. For hybridization, the slides were incubated overnight at 40 °C with hybridization mixture and a final probe concentration of 100 ng/ml. The used oligonucleotide probe (sequence: 5′-TTTAATAACTCGTTATATATATCAGTGTAGCAC-3′) was labelled with digoxigenin at the 3′ end (Eurofins MWG Operon, Ebersberg, Germany). The probe is aimed at 18S rRNA strand and is specific to detect avian Plasmodium spp. [9 (link)]. The digoxigenin-labelled hybrids were detected by incubating the slides with antidigoxigenin-AP Fab fragments (Roche) (1:200) for 1 h at RT. Visualization of the reaction was carried out using the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT) (Roche). Probe specificity has been extensively tested previously [8 (link)]. Tissues from a deceased wild Blackbird Turdus merula free of avian malaria parasites as well as application of an irrelevant oligonucleotide probe (designed for Leishmania spp.) on the experimental samples [see 8 (link)] were used as controls in this study.