Confocal Microscopy ImmunoHistoChemistry and IF
DRAQ5™ shows excellent flexibility as a nuclear DNA stain:
- with fresh tissue (e.g. drosophila embryo), frozen and paraffin embedded sections.
- penetrates deep into tissue to reveal cells below the surface layers
- In ImmunoHistoChemistry (IHC) applications in place of DAPI / PI / Hoechst dyes
- FISH and iFISH applications
DRAQ5™ has been used on many different types of tissue sample.
DRAQ5™ has many benefits for tissue samples. It penetrates deep into tissues, several cell layers deep, and is spectrally compatible with GFP, FITC and most visible range fluorophores. Here are a few examples from the literature:
fresh tissue
Salivary glands excised from gut of Ixodes ticks: glands were teased apart and smeared onto a slide. They were dried and then fixed with acetone prior to staining with antibodies (against B. burgdorferi, Lyme disease) and DRAQ5™ to reveal the host tissue nuclei. (See Grimm et al. PNAS 2004; 101:3142–3147).
Whole allantois explants were fixed in PFA and permeabilised with Triton X-100. Following blocking and labelling with FITC-conjugated antibodies, the tissue was counterstained with DRAQ5™. Complete Z series through the explant were collapsed to show the extent of vascular expansion, based on proximities of nuclei, under different conditions. (See Argraves et al. J Biol Chem 2004; 279 (48):50580-50590 )
frozen sections
Tissue taken from fibrotic lesions in experimental mice were frozen and sliced into 5 µm sections. These were fixed in PFA and peroxidases inactivated with 3% H2O2 in methanol. Sections were incubated with various antibodies (Alexa Fluor 488 & 546 labelled) and after washing DRAQ5™ used to discriminate nucleated cells. (Ishii et al. Stem Cells 2005; 23:699-706).
paraffin-embedded sections
Paraffin-embedded human placenta tissue was cut into 4 µm sections. These were mounted and dried then dewaxed and rehydrated. Following staining with either FITC-labelled antibodies to Annexin II or PP13 the nuclei were counterstained with DRAQ5™ in NaCl/Pi/saponin/BSA buffer. Sections were visualised by confocal microscopy. (See Than et al. Eur J Biochem 2004; 271:1065-1078).
Paraffin-embedded tissue taken from fibrotic lesions in experimental mice were cut into 5 µm sections. These were treated for antigen retrieval. Sections were incubated with various antibodies (Alexa Fluor 488 & 546 labelled) and after washing DRAQ5™ used to discriminate nucleated cells. (Ishii et al. Stem Cells 2005; 23:699-706).
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