New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining.
Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4',6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multi-photon microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.
Universidade Federal do Rio de Janeiro. fabiomg@biof.ufrj.br.
F M
FM
Gomes
I B
IB
Ramos
C
C
Wendt
W
W
Girard-Dias
W
W
De Souza
E A
EA
Machado
K
K
Miranda
D000818:Animals; D002463:Cell Membrane Permeability; D002645:Chickens; D004539:Eimeria; D005453:Fluorescence; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D007211:Indoles; D015388:Organelles / Q000737:chemistry*; D011122:Polyphosphates / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods*; D014349:Trypanosoma cruzi
D005456:Fluorescent Dyes; D007211:Indoles; D011122:Polyphosphates; C007293:DAPI