Background Fluorescence microscopy is a robust tool to study the morphology and function of subcellular compartments or to determine the localization of proteins. signals, demonstrating the necessity to analyze unlabeled cells as unfavorable controls. Introduction Mitochondria from parasitic protists have gained a lot of interest owing to peculiar properties such as RNA editing , citric acid cycle alterations , , apoptotic markers , or mitochondrial proteins import machineries . Therefore, it is essential to stain mitochondria and/or to verify the subcellular localization of protein. Direct fluorescence microscopy, using e.g. green fluorescent proteins (GFP) , and immunofluorescence microscopy (IM) are normal ways of choice for these duties . IM needs (i) a particular major antibody against the proteins appealing and (ii) a reference signal for colocalization. Depending on whether direct or indirect IM is usually applied, either the target-specific primary antibody or an immunoglobulin class-specific secondary antibody has to be fluorescently labeled . The reference signal is usually generated either by staining an established marker protein with a differently fluorescently labeled antibody, by GFP-tagging, or by a fluorescent dye that accumulates at defined subcellular structures . For example, so-called MitoTracker dyes are commonly used to stain mitochondria . Here, we (i) report autofluorescent subcellular structures in promastigotes, (ii) identify the mitochondrion as the source of the autofluorescence, (iii) determine the biophysical properties of the fluorophore promastigotes (10 ml) were cultured in T-flasks in supplemented BHI medium according to standard protocols as previously described , . A thin layer of mid-log phase parasites was decreased on a microscope slide, dried and fixed for 15 min in one of the following solutions: (i) 100% acetone at ?20C, (ii) 20% (v/v) acetone, 80% (v/v) ethanol at ?20C, or (iii) 4% (w/v) paraformaldehyde (PFA) in phosphate buffered saline (PBS) at room temperature. Alternatively, live cell images were recorded after washing the cells three times in 1 ml PBS (1500 g, 15 min, room temperature). The exposure time for the Col13a1 detection of the green fluorescence was 500 ms for fixed and live cell images. For MitoTracker staining, 5106 cells were centrifuged (1500 g, 15 min, room temperature), washed once with 1 ml PBS and resuspended in 1 ml BHI-medium made up of 1 M Mitotracker-Red CM-H2XRos. Promastigotes were stained for 20 min on a shaker at 27C, centrifuged and washed three times with PBS before fixation for 20 min with 4% (w/v) PFA in PBS on a shaker at room heat. After two more washing actions with PBS, cells were centrifuged on cover slips (1500 g, 15 min), Everolimus mounted on microscope slides using Mowiol medium and analyzed the next day using a Zeiss Axiovert 200 M and the software Axiovision. Laser scanning microscopy PFA-fixed promastigotes were further analyzed by laser scanning microscopy at variable excitation wavelengths using a Zeiss LSM780 and the software ZEN 2010. Z-stacks were collected at Z increments of 0.41 m and an excitation wavelength of 458 nm. The same excitation was used to record the emission spectra of whole cells, the cytosol, and the mitochondrion as a non-opisthokont model organism for the extensive analysis of proteins import into all mitochondrial compartments . As the right component of the function, Everolimus we purified four peptide antibodies against different marker protein. Although these antibodies had been perfect for traditional western blot analyses , they didn’t yield satisfactory leads to IM studies, especially because of equivalent fluorescent buildings in unlabeled promastigotes which offered as negative handles. Noteworthy, the fluorescence of such distinctive subcellular buildings in the lack of antibodies (Fig. 1A) had not been only noticed by eye utilizing a selection of cell fixation protocols (Fig. 1B and Strategies), but also without cell fixation (Fig. 1C). Therefore, the fluorescence had not been caused by exterior chemicals, but can be an intrinsic real estate of promastigotes possess distinctive autofluorescent buildings that are detectable with common GFP filtration system sets. Body 1 Recognition of autofluorescent subcellular buildings in promastigotes. Id from the autofluorescent buildings The form and distribution from the autofluorescent structures was Everolimus highly similar to the variable morphology of the single mitochondrion: In dividing promastigotes the mitochondrion has a rather symmetric and circular shape, whereas in non-dividing cells the mitochondrion becomes a single asymmetric tubule . In order to confirm an autofluorescence of the mitochondrion, we subsequently performed a colocalization experiment with a MitoTracker dye. A high degree of colocalization was observed between the autofluorescent signal, detected with the GFP filter set 37, and the MitoTracker signal,.