Confocal Microscopy ImmunoHistoChemistry and IF
DRAQ5™ and CyTRAK Orange™ show excellent flexibility as nuclear counterstains:
- 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™ and CyTRAK Orange™ have been used on many different types of tissue sample.
They have many benefits for tissue samples. They penetrate deep into tissues, several cell layers deep. DRAQ5 and is spectrally compatible with GFP, FITC and most visible range fluorophores. CyTRAK Orange is spectrally compatible with UV, violet-excited dyes, GFP/FITC/AF488 and red-excited dyes. Here are a few examples from the literature:
See more on fixed cells/tissues .. click here
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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