BioProtocols--ICH111

Nuclear DNA staining -- Hoechst 33342 and 33258, DAPI, and Propidium Iodide (PI)

These fluorescent stains are used to primarily label double-stranded DNA and they bind to A-T regions of the DNA helix.

Staining Protocol for Hoechst 33342 and 33258

These probes are UV excited (346nm maximum) and emit maximally at around 460nm (violet/blue). Hoechst 33342 has the advantage of being cell membrane permeant and can, therefore, stain nuclei in living cells as well as fixed cells. Hoechst 33258 (MW 624.0), Hoechst 33342 (MW 615.9).

Stock solution: 2mM (1.2mg/ml) in water or 0.9% NaCl. (One report suggests that at this concentration, it will precipitate if made up in PBS.)

Cell staining Various dilutions (from 1:500 to 1:10,000) in PBS or culture medium have been reported to work.

Incubate for 10 - 15 minutes at room temperature or 37°C.

Rinse 3 - 5 times with PBS or media.

Drain excess buffer and mount in antifade mounting medium.

Reference: Arndt-Jovin et al., J Histochem. Cytochem., 25, 585 - 589, 1977.

Staining Protocol for DAPI

DAPI (4',6-diamidino-2-phenylindole) dihydrochloride (MW = 350.3). Requires UV excitation (maximum excitation = 359nm) and maximum emission is at 461nm (violet/blue). DAPI is quite soluble in water but has limited solubility in phosphate-buffered saline.

Stock solution: 5mg/ml (14.3mM for dihydrochloride salt) in deionized water. May take some time to dissolve in water - sonication may be necessary. Stock solution can be kept at 4°C, protected from light, for at least 6 months. For longer storage, aliquot and freeze at -20°C.

Cell staining:

Dilute stock solution to 300nM in PBS (approx 1ul stock solution in 50ml PBS). (Possible concentrations range from 300nM to 3uM.)

Stain for 1 - 5 minutes.

Rinse sample several times in PBS

Drain excess buffer and mount in antifade mounting medium.

Staining protocol for Propidium Iodide

1. Incubate sample for 20 min at 37C in 2xSSC+100mg/ml RNase.

2. Rinse sections 3x for 1 min each in 2xSSC.

3. Equilibrate sections in 2xSSC.

4. Dilute PI 1/3000 in 2xSSC. Incubate for 1-5 min.

5. Rinse several times with 2x SSC.