New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining

Submitted: 9 July 2013
Accepted: 24 September 2013
Published: 5 November 2013
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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.

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Supporting Agencies

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro,
F.M. Gomes, Universidade Federal do Rio de Janeiro
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem
I.B. Ramos, Universidade Federal do Rio de Janeiro
Laboratório de Bioquímica de Insetos, Instituto de Bioquímica Médica
C. Wendt, Universidade Federal do Rio de Janeiro
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem
W. Girard-Dias, Universidade Federal do Rio de Janeiro
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem
W. De Souza, Universidade Federal do Rio de Janeiro
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem
E.A. Machado, Universidade Federal do Rio de Janeiro
Laboratório de Entomologia Médica, Programa de Biologia Celular e Parasitologia, Instituto de Biofísica Carlos Chagas Filho
K. Miranda, Universidade Federal do Rio de Janeiro
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem

How to Cite

Gomes, F., Ramos, I., Wendt, C., Girard-Dias, W., De Souza, W., Machado, E., & Miranda, K. (2013). New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4’,6-diamidino-2-phenylindole (DAPI)-staining. European Journal of Histochemistry, 57(4), e34. https://doi.org/10.4081/ejh.2013.e34

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