Size-matched preantral follicles were mechanically isolated from mice aged 15 to 16 days as described above. Single follicles were cultured in individual wells in 96 well plates with 1 µm Accell small interfering RNA (siRNA) designed to target exon 8 of the mouse Fshr transcript (GCGAUAACAAUAAUUUGGA; A-062874-17; ThermoScientific). Control follicles were exposed to Accell Nontargeting siRNA (D-001910-01; ThermoScientific) or Accell Red Nontargeting siRNA, containing a DY-547 label (D-001960-01; ThermoScientific). After 24 hours, 10 ng/mL rhFSH was added to all groups, with the exception of an additional control group. Follicles were maintained in culture for another 96 hours and were photographed daily using light microscopy for assessment of growth as described above. Fshr messenger RNA (mRNA) expression was reduced by 50% relative to nontargeting controls (data not shown). Some of the follicles exposed to labeled siRNA were incubated in 5 µm DRAQ5 nuclear stain (Abcam; Cambridge, UK) for 10 min before imaging in chamber slides using a Leica inverted SP5 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany) to visualize penetration of siRNA into the follicle.
Draq5 nuclear stain
Manufactured by Abcam
Sourced in United Kingdom
DRAQ5 is a cell-permeable fluorescent dye that selectively stains the nuclei of cells. It is a far-red fluorescent DNA dye with excitation and emission maxima at 647 nm and 670 nm, respectively. DRAQ5 can be used to label and visualize nuclei in living or fixed cells for a variety of applications, including flow cytometry, microscopy, and high-content screening.
Automatically generated - may contain errors
3 protocols using draq5 nuclear stain
1
Silencing Fshr via Accell siRNA in Preantral Follicles
2
Immunofluorescence Staining of Primary Urinary Cells
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Primary urinary cells were grown in a 96-well plate coated with 0.1% Gelatine (MilliporeSigma) until confluent. After treatment with vehicle/lyso-Gb3, cells were washed in PBS, fixed in 4% paraformaldehyde, washed again in PBS, and blocked/permeabilized in 5% BSA, 0.1% Triton X-100 in PBS for 1 hour at room temperature. After 3 washing steps in PBS, cells were stained with primary antibody (1:200; Sigma-Aldrich, HPA005459) overnight. The next day, after 3 additional washing steps in PBS, cells were incubated in secondary antibody (1:200; LI-COR IRDye 800cw) and Draq5 nuclear stain (1:1,000; Abcam, 108410) for 1 hour at room temperature. Cells were washed again 3 times and imaged with a LI-COR Odyssey imager at 3 μm focus, 89 μm resolution.
3
Visualizing Venus Fluorescent Protein in H4 Neuroglioma Cells
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Human H4 neuroglioma cells cultured in glass-bottom dishes were transfected with Venus fragments as described above using Turbofect transfection reagent. Transfected H4 cells were stained with 5 µM DRAQ5 nuclear stain (ab108410, Abcam) and incubated at 37 °C for 5 min prior to cell imaging. Live cell imaging was performed on cells maintained in a humidified incubator (37 °C, 5% CO2) using a Zeiss LSM 710 microscope. To obtain large field of view for analysis, images were collected using a 4 × 4 tiles with a 20 × Fluar 0.8 NA (Carl Zeiss, Jena, Germany) at 1024 × 1024 pixels. Excitation of Venus fluorescent protein (VFP) was carried out with 514-nm argon laser while DRAQ5 was excited at 633 nm with a HeNe laser. Emitted light was collected through appropriate filters to eliminate any spill-overs. Image acquisition and processing were carried out using ZEN 2.1 software (Carl Zeiss, Jena Germany).
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