A modified fluorescence in situ hybridization protocol for Plasmodium falciparum greatly improves nuclear architecture conservation

Abstract

Fluorescence in situ hybridization (FISH) has been used extensively in the study of nuclear organization and gene positioning in Plasmodium falciparum. While performing FISH with published protocols, we observed large variations in parasite nuclear morphology. We hypothesized that these inconsistencies might be due to the type of parasite preparation prior to FISH, which commonly involves air-drying, prompting us to develop a new fixation protocol. Here we show both qualitatively and quantitatively that compared to air-dried and briefly fixed parasites, longer fixation in suspension leads to improved conservation of nuclear structure and lower intra-population variation of nuclear shape as well as area after FISH development. While the fixation protocol per se does not cause detectable disruptions in nuclear morphology, it greatly influences the conservation of nuclear shape and size during the most stringent steps of FISH. The type of fixation used also influences the detection of telomeric clusters, and we show that the new fixation protocol permits improved conservation of the chromosome end cluster perinuclear distribution and higher colocalization indexes for two adjacent chromosome end probes, Rep20 and telomere. Overall, the results indicate that our alternative protocol dramatically improves conservation of the nuclear architecture compared to previously reported Plasmodium DNA-FISH protocols and highlights the necessity of carefully choosing the fixation protocol for FISH.