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DAPI STAINING PROTOCOL

DAPI 4,6 diamino-2-phenylindole 2HCl
DAPI staining is specific at pH 7, at other pH non-specific staining occurs.

1000X DAPI stock

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dissolve 0.2 mg DAPI in 1 ml dist. H2O.

store at 4C in dark

McIlvaines buffer pH 7.0

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stock solution A: 0.1 M citric acid

stock solution B: 0.2 M Na2HPO4

for 200 ml use 4.2 g

for 800 ml use 22.7 g

Mix 2 parts of stock A and 8 parts of stock B to make pH 7.0 buffer.

DAPI staining solution (600 nM DAPI)

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10 ml McIlvaines pH 7.0
10 l 1000X DAPI stock

Staining of paraffin embedded sections:

Use Coplin jars for the following steps until DAPI staining. Dewax sections in xylene (2 x 5
min), rehydrate in ethanol series (absolute, 95% for 5 min, 70%, 30% ethanol, dH2O for 3 min),
equilibrate in McIlvaines buffer (5 min, prepared fresh). Drain slides, put them on paper towel,
apply DAPI staining solution on slides (~200 l), incubate 15 min in dark (cover with a box),
drain excess solution, mount coverslips with Gel Mount (or other aqueous mounting medium).
Observe with UV excitation, nuclei will emit cold blue fluorescence.

Counterstaining:
Gel Mount DAPI
- 1 ml Gel Mount
- 1 l 1000X DAPI stock
Mix thoroughly, but take care not to create too much bubbles.
Use DAPI staining as a final step in any fluorescent staining procedure. If the final wash is in a
buffer with a pH close to 7, such as PBS, equilibration is not necessary, otherwise equilibrate in
McIlvaines buffer for 5 min. Drain excess solution from the slide. Apply two separate drops of
Gel Mount DAPI on the sections, then carefully lower the coverslip on sections. Let stand in
dark for few minutes, then observe DAPI stain with UV excitation.