Confocal Microscopy fixed cells / nuclei
DRAQ5™ and CyTRAK Orange™ are completely compatible with different fixation strategies including paraformaldehyde (PFA), methanol and acetone.
Permeabilisation of cells to allow staining of intracellular antigens does not impact on subsequent DRAQ5™ or CyTRAK Orange™ staining.
Using formaldehyde as fixative - fixation (& permeabilisation) then DRAQ5™ / CyTRAK Orange™:
Maiuri et al. J. Immunol. 2008;180:7697-7705
CyTRAK Orange™ used as nuclear counterstain for confocal microscopy: Adherent cells (primary bronchial polyp mucosa, and epithelial cell lines IB3-1, C38) were fixed with formaldehyde, permeabilized and stained with antibodies and CyTRAK Orange™. The nuclear counterstain is proved to be compatible with FITC in IF giving clear delineation of nuclei.
Foley et al. Frontiers in Bioscience 2005; 10:1302-1312
To show endocytosis inhibitor effects on actin organisation, following inhibitor treatment, VSMCs were fixed in formaldehyde, permeabilised in Triton-X100 and stained with FITC conjugated anti-actin antibodies. Cells were then stained with DRAQ5™ to reveal the nuclei.
Yu et al. Molec Biol Cell 2005; 16:433-445
To explore the mechanisms of polarity in epithelial cells a monolayer of MDCK cells was overlaid with collagen gel. After a time the gel was removed, the cells fixed in formaldehyde, blocked and permeabilised with a gelatin/PBS/saponin buffer and stained with Alexa Fluor 532 & 488 conjugated antibodies. Finally, cells were stained with DRAQ5™ (1:1000 dilution, 37°C, 5 min.). Similar procedures were carried out for cysts cultured in collagen gel. Different inhibitors were applied to disrupt the polarisation.
Pelkmans et al. Nature 2005; 436:78-86
Endocytotic ligand trafficking was studied by high content assays on the Opera (Evotec Technologies). AlexaFluor 488- and DiI-labelled ligands were applied and HeLa cells incubated for 10 - 60 minutes. Some cells were stably transfected with Cav1-GFP. The distribution of Cav1 and the ligands was followed under different siRNA gene silencing. Cells were fixed (formaldehyde) to stop further endoytosis and stained with DRAQ5™.
Using formaldehyde as fixative AFTER staining with DRAQ5™:
Mitta et al. Nucl. Acids Res. Online 2002; 30 (21):e113
Lentivirus-transduced CHO cells were stained with DRAQ5™. Following fixation with PFA they were imaged for YFP reporter gene expression as a measure of efficiency of tranduction relative to parental strains. Both confocal and conventional fluorescence microscopy was used.
Using acetone as fixative:
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).
Using methanol as fixative:
Nuclei & Oocytes:
Schmitt et al. FEBS Letters 2002; 518: 23-28
Xenopus half oocytes & oocyte nuclei were fixed in methanol (-80°C, 60 min.), rehydrated in PBS, stained with anti-phospho-Histone antibodies (AlexaFluor 488) and mounted in PBS/DRAQ5 2.5 µM. Phosphorylation of Histone H3 was found to be insufficient alone for chromosome condensation during meiotic maturation.
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