Cover Image

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

F.M. Gomes, I.B. Ramos, C. Wendt, W. Girard-Dias, W. De Souza, E.A. Machado, K. Miranda
  • F.M. Gomes
    Universidade Federal do Rio de Janeiro, Brazil
  • I.B. Ramos
    Universidade Federal do Rio de Janeiro, Brazil
  • C. Wendt
    Universidade Federal do Rio de Janeiro, Brazil
  • W. Girard-Dias
    Universidade Federal do Rio de Janeiro, Brazil
  • W. De Souza
    Universidade Federal do Rio de Janeiro, Brazil
  • E.A. Machado
    Universidade Federal do Rio de Janeiro, Brazil

Abstract

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.

Keywords

DAPI, polyphosphate, fluorescence, fluorimetry

Full Text

PDF
HTML
Submitted: 2013-07-09 23:33:16
Published: 2013-11-05 11:30:08
Search for citations in Google Scholar
Related articles: Google Scholar
Abstract views:
2122

Views:
PDF
775
HTML
485

Article Metrics

Metrics Loading ...

Metrics powered by PLOS ALM


Copyright (c) 2013 F.M. Gomes, I.B. Ramos, C. Wendt, W. Girard-Dias, W. De Souza, E.A. Machado, K. Miranda

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
 
© PAGEPress 2008-2017     -     PAGEPress is a registered trademark property of PAGEPress srl, Italy.     -     VAT: IT02125780185