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.