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Revid: 05/17/99
Product Information Propidium Iodide Nucleic Acid Stain
P-1304propidium iodide
P-3566
propidium iodide, 1 mg/mL solution in water
Introduction
Propidium iodide (PI) binds to DNA by intercalating between the bas with little or no quence preference and with a stoichi-ometry of one dye per 4–5 ba pairs of DNA.1 PI also binds to RNA, necessitating treatment with nucleas to distinguish be-tween RNA and DNA staining. Once the dye is bound to nucleic acids, its fluorescence is enhanced 20- to 30-fold, the fluores-cence excitation maximum is shifted ~30–40 nm to the red and the fluorescence emission maximum is shifted ~15 nm to the blue.2 Although its molar absorptivity (extinction coefficient) is relatively low, PI exhibits a sufficiently large Stokes shift to allow simultaneous detection of nuclear DNA and fluorescein-labeled antibodies, provided the proper optical filters are ud.Propidium iodide is suitable for fluorescence microscopy, confo-cal lar scanning microscopy, flow cytometry and fluorometry.PI is membrane impermeant and generally excluded from viable cells. PI is commonly ud for identifying dead cells in a
population and as a counterstain in multicolor fluorescent tech-niques. The counterstaining protocols below are compatible with a wide range of cytological labeling techniques — direct or indi-rect antibody-bad detection methods, mRNA in situ hybridiza-tion or staining with fluorescent reagents specific for cellular structures. The protocols can be modified for tissue staining.
Materials
prepareforStorage and Handling
Upon receipt, store the solid (P-1304) at room temperature,protected from light. The solid should be stable for at least a year. Store the solution of PI (P-3566) at 4°C, protected from light. To make a stock solution from the solid form, dissolve PI (MW = 668.4) in deionized water (dH 2O) at 1 mg/mL (1.5 mM)and store at 4°C, protected from light. When handled properly,solutions are stable for at least 6 months.
Caution : PI is a known mutagen. Solutions containing PI should be poured through activated charcoal before disposal.The charcoal must then be incinerated to destroy the dye.
Fluorescence Spectral Characteristics
When bound to nucleic acids, the absorption maximum for PI is 535 nm and the fluorescence emission maximum is 617 nm (Figure 1). PI can be excited with a xenon or mercury-arc lamp or with the 488 line of an argon-ion lar. Molecular Probes offers high quality Omega Optical filter ts for fluorescence microscopy. Filter ts O-5725 and O-5729 are optimized to match the spectral properties of PI. Generally, PI fluorescence is detected in the FL2 channel of flow cytometers.
Protocol for Counterstaining Adherent Cells for Fluorescence Microscopy
Sample Preparation
U the fixation protocol appropriate for your sample. PI stain-ing is normally performed after all other staining. Note that per-meabilization of the cells is required for counterstaining with PI.
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RNa Treatment
RNa treatment is required if samples are fixed in paraform-aldehyde, formaldehyde or glutaraldehyde. If samples are fixed with methanol/acetic acid or acetone, RNa treatment is usually not required.
1.1 Equilibrate the sample briefly in 2X SSC (0.3 M NaCl,
0.03 M sodium citrate, pH 7.0).
Figure 1. Absorption and fluorescence emission profiles of propidium iodide bound to dsDNA.
1.2 Incubate in 100 µg/mL DNa-free RNa in 2X SSC for
20 minutes at 37°C.
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1.3 Rin samples 3 times, 1 minute each, in 2X SSC. Counterstaining Protocol
2.1 Equilibrate the sample in 2X SSC.
2.2 Make a 500 nM solution of PI by diluting the 1 mg/mL (1.5 mM) stock solution 1:3000 in 2X SSC. About 300 µL is usually enough stain for one coverslip preparation. Incubate cells, covered with the dilute stain, for 1–5 minutes.
2.3 Rin samples veral times in 2X SSC. Drain excess buffer from coverslip and mount in a medium with an antifade reagent such as provided in Molecular Probes’ SlowFade®Antifade Kit, SlowFade Light Antifade Kit or ProLong® Antifade Kit.
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2.4 View sample using a fluorescence microscope with appropri-ate filters (e Fluorescence Spectral Characteristics).
Protocol for Counterstaining Cells in Suspension for Flow Cytometry
Sample Preparation
U the fixation protocol appropriate for your sample, or u the following protocol.
3.1 Collect a volume of cell suspension corresponding to 2 × 105 to 1 × 106 cells. Pellet the cells by centrifugation. Discard the supernatant, tap the tube to resuspend pellet in the residual liquid and add 1 mL of phosphate-buffered saline (PBS) at room tem-perature.
3.2 Transfer the full volume of resuspended cells to 4 mL of absolute ethanol at -20°C by pipetting the cell suspension slowly into the ethanol while vortexing at top speed. Leave in ethanol at -20°C for 5 to 15 minutes.
3.3 Pellet the cells by centrifugation, discard the ethanol, tap the tube to loon the pellet and add 5 mL PBS at room temperature. Allow cells to rehydrate for 15 minutes. Counterstaining Protocol
4.1 Make a 3 µM solution of PI by diluting the 1 mg/mLbypasd
(1.5 mM) stock solution 1:500 in Staining Buffer (100 mM Tris,pH 7.4, 150 mM NaCl, 1 mM CaCl
2
, 0.5 mM MgCl
2
, 0.1% Nonidet® P-40). A 1 mL volume will be required for each cell sample.
4.2 Centrifuge the cell suspension from step 3.3, discard the supernatant, tap to loon the pellet and add 1 mL of PI-Staining Buffer. Incubate for 15 minutes at room temperature and analyze by flow cytometry in the prence of the dye. If the cells are to be viewed by fluorescence microscopy, centrifuge the sample, re-move the supernatant and resuspend the cells in fresh buffer. Apply a drop of the suspension to a microscope slide, cover with a coverslip and view.
Protocol for Chromosome FISH Counterstaining Sample Preparation
Prepare the specimen according to standard procedures.3,4 Briefly rin the final preparations in dH
2
O before counter-staining to remove residual buffer salts from the slide. Air dry. This final rin will help reduce nonspecific background staining on the glass.
Counterstaining Protocol
5.1 Make a 1.5 µM PI staining solution by diluting the 1 mg/mL (1.5 mM) stock solution 1:1000 in PBS. Pipet 300 µL of this staining solution directly onto the specimen. If necessary, RNa
A (freshly made) may be added to a final concentration of
10 µg/mL. A plastic coverslip can be ud to distribute the dye evenly on the slide.
5.2 Incubate the specimen in the dark for 30 minutes at room temperature, or at 37°C if RNa is included.
5.3 Remove the coverslip and rin briefly with PBS or dH
2
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O to remove unbound dye.
5.4 Remove excess liquid from the slide by gently blotting around the sample with an absorbent tissue. Place a glass cover-slip on the slide, and al the edges with wax or nail polish. Alternatively, the preparation can be mounted in an antifade re-agent according to the manufacturer’s directions.
5.5 View sample using a fluorescence microscope with appropri-ate filters (e Fluorescence Spectral Characteristics).
wild wild westReferences
1. J Mol Biol 13, 269 (1965);
2. Meth Cell Biol 30, 417 (1989);
英语培训中心3. Meth Enzymol 168, 741 (1989);
4. Pardue, M.L. in Nucleic Acid Hybridization, A Practical Approach,. B.D. Hames and S.J. Higgins, Eds., IRL Press, Oxford, England (1985).
Product List Current prices may be obtained from our Web site or from our Customer Service Department.
Cat #Product Name Unit Size P-7481ProLong® 1 kit P-1304pro pidium 100 mg P-3566propidium iodide *1.0 mg/mL solution in water*........................................................................................................................10 mL S-2828SlowFade®
.......................................... 1 kit S-7461SlowFade®Light . 1 kit
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