Table 8.4 Properties of classic nucleic acid stains.

Cat. No.DyeEx/Em *MW †Fluorescence Emission ColorApplications ‡
A666Acridine homodimer431/498685.69Green
  • Impermeant
  • AT-selective
  • High-affinity DNA binding
A1301
A3568 §
Acridine orange

500/526 (DNA)
460/650 (RNA)

301.82
301.82
Green/Red
  • Permeant
  • RNA/DNA discrimination measurements
  • Lysosome labeling
  • Flow cytometry
  • Cell-cycle studies
A13107-AAD (7-amino-actinomycin D)546/6471270.45Red
  • Weakly permeant
  • GC-selective
  • Flow cytometry
  • Chromosome banding
A7592Actinomycin D4421255.43None
  • Chromosome banding
A1324ACMA419/483258.71Blue
  • AT-selective
  • Alternative to quinacrine for chromosome Q banding
  • Membrane phenomena
D1306
D3571
D21490
DAPI358/461350.25
457.49
350.25
Blue
  • Semi-permeant
  • AT-selective
  • Cell-cycle studies
  • Mycoplasma detection
  • Chromosome and nuclei counterstain
  • Chromosome banding
D1168
D11347
D23107
Dihydroethidium518/605

315.42
315.42
315.42

Red **
  • Permeant
  • Blue fluorescent until oxidized to ethidium
15585-011Ethidium bromide518/605394.31Red
  • Impermeant
  • dsDNA intercalator
  • Dead-cell stain
  • Chromosome counterstain
  • Electrophoresis
  • Flow cytometry
  • Argon-ion laser excitable
E1169Ethidium homodimer-1 (EthD-1)528/617856.77Red
  • Impermeant
  • High-affinity DNA labeling
  • Dead-cell stain
  • Electrophoresis prestain
  • Argon-ion and green He-Ne laser excitable
E3599Ethidium homodimer-2 (EthD-2)535/6241292.71Red
  • Impermeant
  • Very high-affinity DNA labeling
  • Electrophoresis prestain
E1374Ethidium monoazide464/625
(unbound) ††
420.31Red
  • Impermeant
  • Photocrosslinkable
  • Compatible with fixation procedures
H7593Hexidium iodide518/600497.42Red
  • Permeant, except gram-negative bacteria
  • Stains nuclei and cytoplasm of eukaryotes and some bacteria
H1398
H3569 §
H21491
Hoechst 33258 (bis-benzimide)352/461623.96
623.96
623.96
Blue
  • Permeant
  • AT-selective
  • Minor groove–binding
  • dsDNA-selective binding
  • Cell-cycle studies
  • Chromosome and nuclear counterstain
H1399
H3570 §
H21492
Hoechst 33342350/461615.99
615.99
615.99
Blue
  • Permeant
  • AT-selective
  • Minor groove–binding
  • dsDNA-selective binding
  • Cell-cycle studies
  • Chromosome and nuclear counterstain
H21486Hoechst 34580392/498560.96Blue
  • Permeant
  • AT-selective
  • Minor groove–binding
  • dsDNA-selective binding
  • Cell-cycle studies
  • Chromosome and nuclear counterstain
H22845Hydroxystilbamidine385/emission varies with nucleic acid472.53Varies
  • AT-selective
  • Spectra dependent on secondary structure and sequence
  • RNA/DNA discrimination
  • Nuclear stain in tissue
L7595LDS 751

543/712 (DNA)
590/607 (RNA)

471.98Red/infrared
  • Permeant
  • High Stokes shift
  • Long-wavelength spectra
  • Flow cytometry
N21485Nuclear yellow355/495651.01Yellow
  • Impermeant
  • Nuclear counterstain
P1304MP
P3566 §
P21493
Propidium iodide (PI)530/625668.40
668.40
668.40
Red
  • Impermeant
  • Dead-cell stain
  • Chromosome and nuclear counterstain
* Excitation (Ex) and emission (Em) maxima in nm. All excitation and emission maxima were determined for dyes bound to double-stranded calf thymus DNA in aqueous solution, unless otherwise indicated. † Molecular weight (MW) for the anhydrous compound. ‡ Indication of dyes as "permeant" or "impermeant" are for the most common applications; permeability to cell membranes may vary considerably with the cell type, dye concentrations and other staining conditions. § Available in aqueous solution for those wishing to avoid potentially hazardous and mutagenic powders. ** After oxidation to ethidium. †† Prior to photolysis; after photolysis the spectra of the dye/DNA complexes are similar to those of ethidium bromide–DNA complexes.