2.9. Deposition of aluminium on Spongilla lacustris,Euglena gracilis and Dictyostelium discoideum studied by lumogallion staining.
E. gracilis cells were killed by liquid nitrogen freezing, transferred to glass slides and dried briefly to adhere to the glass surface. S. lacustris were processed in small Petri dishes. The culture of D. discoideum with unicellular amoebae and different multicellular stages (mounds, sorogens, and fruiting bodies) was washed off from the agar plate with distilled water and transferred onto glass chamber slides. The cells and multicellular structures representing different developmental stages were killed with sodium azide and left to air dry briefly to adhere to the bottom of the glass chamber. Sodium azide was removed by rinsing with distilled water.
Treatment with aluminium was carried out by incubation with 1 mM solution of alum (KAl(SO4)2) in distilled water at room temperature for 30 min. The alum solution was removed, the specimens on the glass slides or in Petri dishes were rinsed 5 times with distilled water to remove unbound aluminium, fixed in 2% paraformaldehyde (PFA) for 20 min, and rinsed 2 times with distilled water to remove PFA.
The presence of bound aluminium was detected with standard lumogallion (5-chloro-3[(2,4-dihydroxyphenyl)azo]-2-hydroxybenzenesulfonic acid) staining, described elsewhere and used to detect aluminium at low (around 2μM) concentrations (Kataoka et al., 1997; Mold et al., 2014). The samples were stained in 0.025 mM lumogallion in 0.1 M sodium acetate buffer pH 5.2 at 50 °C for 50 min in the dark. The lumogallion solution was removed, and the stained preparations were rinsed with distilled water. In the case of D. discoideum, samples were also counterstained with the DNA dye Hoechst 342 in a conventional manner to visualize the positions of cell nuclei. Prepared samples covered with a cover-glass were immediately examined under a Leica DMR Fluorescent Microscope with an UV excitation and emission at 488 nm (green, characteristic for lumogallion-Al complex); colonial amoebae were also examined at 408 nm (blue, Hoechst-DNA). E. gracilis preparations were also examined under the confocal fluorescent microscope Leica TCS SP5 at UV excitation and 488 nm emission to reveal cellular localization of the fluorescent structures. Each sample was prepared in triplicate.
In the cell autofluorescence controls, both incubation in alum and lumogallion staining steps were omitted. The lumogallion non-specific binding controls (”Al-negative”) lacked the alum incubation step.