Immunofluorescence staining protocol for STED nanoscopy of Plasmodium-infected red blood cells

Abstract

Immunofluorescence staining is the key technique for visualizing organization of endogenous cellular structures in single cells. Labeling and imaging of blood stage Plasmodium falciparum has always been challenging since it is a small intracellular parasite. The gold standard for parasite immunofluorescence is fixation in suspension with addition of minute amounts of glutaraldehyde to the paraformaldehyde-based solution. While this maintains red blood cell integrity, it has been postulated that antigenicity of the parasite proteins was, if at all, only slightly reduced. Here we show the deleterious effect that even these small quantities of glutaraldehyde can have on immunofluorescence staining quality and present an alternative cell seeding protocol that allows fixation with only paraformaldehyde. The highly improved signal intensity and staining efficiency enabled us to carry out RescueSTED nanoscopy on microtubules and nuclear pores and describe their organization in greater detail throughout the blood stage cycle.

CRISPR/Cas9-engineered inducible gametocyte producer lines as a novel tool for basic and applied research onPlasmodium falciparummalaria transmission stages

“…S5). Our results on asexual parasites recapitulate previous findings obtained by high-resolution microscopy 80,84,85 . These studies showed that merozoites and ring stage parasites possess only three to seven nuclear pores that are closely clustered in one region of the nuclear membrane.…”

“…S4). Validation of the NF54/iGP2_NUP313-mSc line by live cell fluorescence imaging verified the expected localization of nuclear pores across the different IDC stages as determined in previous studies 80,84,85 (Fig. 4a).…”

“…As ring stage parasites develop into trophozoites the number of NPCs increases and up to 60 NPCs are evenly distributed throughout the nuclear envelope. During schizogony the number of pores per nucleus decreases with increasing numbers of nuclei formed, such that in late schizonts each nucleus of the developing merozoites again possesses only a small number of clustered nuclear pores 80,84,85 . The functional relevance of this intriguing redistribution of NPCs is currently unknown, but it may be linked to regulatory strategies of stage-specific gene expression during the IDC and/or the coordination of nuclear segregation during schizogony 84 .…”

CRISPR/Cas9-engineered inducible gametocyte producer lines as a novel tool for basic and applied research on Plasmodium falciparum malaria transmission stages

“…S5). Our results on asexual parasites recapitulate previous ndings obtained by highresolution microscopy 80,84,85 . These studies showed that merozoites and ring stage parasites possess only three to seven nuclear pores that are closely clustered in one region of the nuclear membrane.…”

“…Validation of the NF54/iGP2_NUP313-mSc line by live cell uorescence imaging veri ed the expected localization of nuclear pores across the different IDC stages as determined in previous studies 80,84,85 ( Fig. 4a).…”