Monday, 27 March 2017

Counterstaining FFPE sections in the far-red

We’ve had recent questions about using DRAQ5™ with formalin-fixed paraffin-embedded (FFPE) tissue sections.  In response, we’ve prepared a review of the technology status:

In short, DRAQ5™ does work on FFPE samples and has been demonstrated in peer-reviewed publications.  We do not provide a dedicated protocol for FFPE sections in our technical datasheet as the processing of the sections is upstream of the use of DRAQ5™ which is last of all, prior to imaging.  Also, the pre-treatment of sections may differ according to the determinants being probed but these treatments do not impact on DRAQ5™ as long as the integrity of nuclear DNA is unaffected.  We do, however, have a suitable protocol (for fixed cells) and DRAQ5™ is already cited in countless papers for this (the majority of the ca. 5500 to date).

With FFPE sections it is imperative to perform de-waxing and antigen retrieval procedures and those remain unchanged by switching to DRAQ5™.  The key consideration is that once the sample is amenable to antibody probing it is more than ready for DRAQ5™ counterstaining.

A search of the published literature provides two useful examples that include clear and comprehensive methods sections on FFPE and staining with DRAQ5™:

Kessler et al.  Am J Pathol 2012, 180:186–198
Authors use DRAQ5™ at 6.25 µM (as counterstain)

Fleskens, et al, Histopathology, 2010 57: 14–26
Authors use DRAQ5™ at 25 µM (as counterstain and measure of ploidy)

Fleskens et al. are part of a clinical histopathology service (Nijmegen, NL) and use DRAQ5™ to measure ploidy, requiring its stoichiometric labeling capability and hence the higher concentration used to achieve this.

These papers would suggest using DRAQ5™at a concentration of between 6 and 25 µM* (1:800 – 1:200 dilution of the standard 5 mM product, e.g. DR50200) depending on the information required; simple counterstaining to ploidy estimations.  We strongly recommended NOT washing sections after DRAQ5™ staining.  If necessary, excess liquid can be tamped away with an absorbent, lint-free tissue prior to mounting with a coverslip.**  Besides its spectral properties, a major advantage of DRAQ5™is its convenience: it has excellent shelf-life and is ready-to-use straight from the fridge in an aqueous solution.

A recent, compelling paper by Elfer et al. (PLoS One 2016) compares the use of DRAQ5™ in place of Hematoxylin in histochemical procedures, giving equivalent results, starting first on FFPE and then moving to an accelerated procedure compatible with intra-operative timescales which H&E cannot deliver.

Elfer et al. use a higher concentration as they are developing an accelerated procedure and are aiming for a SIM-based approach for rapid intra-operative investigation and reporting.

Altogether, these confirm that DRAQ5™ performs as a counterstain for FFPE sections.

Visit our website for all the technical and ordering information for DRAQ5™and find out for yourself why people choose DRAQ5 for their FFPE sections!

*Our recommended protocol for the counterstaining of fixed cell preparations calls for a DRAQ5™ concentration of 5 µM, and this is reflected in the majority of the publications and represents a good starting point.
**To mount sections with a coverslip we would recommend the use of Prolong® Gold (Thermo Fisher Scientific) or Fluoromount-G® (Southern Biotech Associates, Inc.).  It is essential that the mountant does NOT already contain a counterstain such as DAPI.