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.

Activation and clustering of a Plasmodium falciparum var gene are affected by subtelomeric sequences

“…Indirect DNA FISH was performed using a modification of an existing method . Roche Hi Prime biotin and digoxigenin labelling kits were used to random prime label 1 μg of purified dsDNA (rep20 or var2csa ) as per the manufacturer's method.…”

Epigenetic function of Granulocytic nuclei? Designing a new line of research

“…All of the experiments described in this paper need a way of combining an in situ hybridisation with nuclear staining and a practically perfect nuclear shape conservation. The deformation is most likely caused by fixation and more efficient fixation techniques can help solving the problem completely or partially (Contreras-Dominguez et al 2010), but also the high temperatures needed for the separation of DNA strands can play a role in it and we may have to face this problem in order to follow this research line.…”

In Situ Fluorescence Visualization of Transcription Sites and Genomic Loci in Blood Stages of Plasmodium falciparum

“…Plasmodium immunofluorescence and FISH preparations are generally examined using wide-field microscopes (1-6, 9-13), though confocal microscopy has also been applied with success (14). Since the parasite nucleus, in the asexual ring stage, is only 1.5-2 μm in diameter, the objective magnification recommended is 100×.…”

“…The free-parasite fixation in suspension has been shown to improve conservation of the nuclear shape and size in Plasmodium parasites (14). Moreover, the suspension of fixed parasites can be stored at 4°C up to 2 months.…”