DAPI staining ...¢ [?Δ? ]
040813 yamaichi

A. Fixed on slide glass

1.1 Centrifuge 2 ml of growing culture at 6,000 rpm for 3 min.
Pour off the sup and resuspend the pellet with remaining small amount (~50 μl) of broth.

1.2 Drop 10-20 μl of cell suspension on a poly-L-Lysine coated glass slide.

1.3 Keep slides until culture is dried (it takes several minutes).
If necessary, air-dry with blower (by cool air).

1.4 Drop 1 ml of 100% Methanol to cover the slide. Keep 3 min.

1.5 Pour off the methanol.
Rinse the slide by D.W. with wash bottle.

1.6 Air-dry with blower (by cool air).
While drying, incubate the mounting solution at 37?C.

1.7 Drop 5 μl of the pre-warmed mounting solution onto a slide where cells are fixed.

1.8 Put a cover glass.

B. Fixed at sample tube

1.1 Transfer 1 ml of growing culture into 15-ml centritube.
Add 9 ml of 80% Methanol. Keep 10 min.

1.2 Centrifuge the tube at 3,000 rpm for 10 min.
Pour off the sup and resuspend the pellet with remaining small amount (~0.1 ml) of methanol solution.

1.3 Drop 10-20 μl of cell suspension on a poly-L-Lysine coated glass slide.

1.4 Keep slides until culture is dried (it takes few minutes).

1.5 Rinse the slide by D.W. with wash bottle.

1.6 Air-dry with blower (by cool air).
While drying, incubate the mounting solution at 37?C.

1.7 Drop 5 μl of the pre-warmed mounting solution onto a slide where cells are fixed.

1.8 Put a cover glass.

Reagents

1. mounting solution
90% Glycerol Mix 1:1
1 mg/ml p-phenylenediamine dihydrochloride 0.3 mg/ml DAPI (with DABCO) in glycerol
0.15 mg/ml DAPI 0.2% p-phenylenediamine in [2xPBS, 80% glycerol]
1x PBS

store at -20?C