Standard immunofluorescence staining Procyclic and promastigote cells were washed and re-suspended in PBS to 1 1 107 cells ml?1


Standard immunofluorescence staining Procyclic and promastigote cells were washed and re-suspended in PBS to 1 1 107 cells ml?1. [2]. Another prominent feature is the presence of the flagellum, the principal motility organelle, which contains the evolutionarily conserved microtubule-based axoneme, and the kinetoplastid-specific paraflagellar rod [2]. The flagellum specifies, via association with 5(6)-FITC its base, the position of single-copy organelles, such as the kinetoplast (i. e. mitochondrial DNA) and the flagellar pocket [3]. Moreover, in [14] and [15], as well as the recent study of a mitochondrial genome segregation factor in [16], demonstrated that due to small sizes of their cells ExM offers clear opportunities for analysing structural features of these important parasites. Moreover, due to a well-defined morphology of their cellular cytoskeleton protists enable assessing the ExM approach at cellular dimensions. In this work, we set out to optimize and validate ExM for and cells, which possess a complex, highly defined and relatively well-characterized microtubule-based cytoskeleton, as described above. We show that data obtained by ExM recapitulate the previous EM observations with regard to the presence, positioning, orientation and shape of individual structures. However, caution should be observed when using ExM to determine distances, as individual structures may expand to a different extent. This was previously noticed in other experimental systems (e.g. [17,18]). Importantly, we observed that ExM enables resolving individual cytoskeletal components, such as the microtubules. This, together with the quantitative aspect of the approach, facilitated a better understanding of less well-appreciated structures, such as the neck microtubule 5(6)-FITC cells short microtubules or plate-like structures associated with basal and pro-basal bodies. Moreover, ExM facilitated studies of FLJ21128 rare cell types in a population, providing detailed information on the structure of the mitotic spindle and cleavage furrow in cells, which naturally 5(6)-FITC occur in the tsetse fly midgut. They can, however, be readily cultured in a laboratory, and hence their morphology, cellular architecture and changes to these during the cell cycle have been extensively characterized [5,7]. We employed a variation of ExM termed ultrastructure ExM, which is based on fixation of cells with 4% formaldehyde and 4% acrylamide, followed by gelation with 19% sodium acrylate, 10% acrylamide and 0.1% N, N’-methylenebisacrylamide, denaturation with 200 mM sodium dodecyl sulfate at 95C, and antibody staining [10]. Following a described protocol for handling free-swimming cells [10], we adhered procyclic cells to a glass coverslip and further processed for ExM. To visualize expanded microtubule-based structures to validate the procedure, we tested several anti-tubulin antibodies: the polyclonal rabbit anti-alpha tubulin antibody (table?1 for specification), the mouse monoclonal anti-alpha tubulin antibody TAT1 [19], the mouse anti-beta tubulin antibody KMX-1 [20] and the mouse monoclonal anti-acetylated tubulin antibody C3B9 [19]. Table?1. List of used antibodies. ExMexpansion microscopy; IFimmunofluorescence. cell fixed on a coverslip and stained with the rabbit polyclonal anti-tubulin antibody. (i) En face view; (ii) side view. Axes labels are in m for all three-dimensional reconstructed images. (cell fixed on a coverslip and stained with the C3B9 antibody. (i) en face view; (ii) side view. (cell in cytokinesis, which was fixed on a coverslip and stained with the C3B9 antibody. (i) En face view; (ii) side view. old Aold axoneme; new Anew axoneme. (cell in cytokinesis, which was fixed in solution before being settled on a coverslip and stained with the C3B9 antibody. 5(6)-FITC (i) En face view; 5(6)-FITC (ii) side view. (cell stained with the C3B9 antibody. (cell with a single flagellum stained with the C3B9 antibody..