Fluorochrome banding
For fluorchrome banding with 4',6-Diamidin-2-phenyl-indol (DAPI)
polytene chromosomes have been incubated for 10 min.
In order to get a differential fluorochrome banding of blue, pink and white
preparations have been stained in a mixture of DAPI and propidiumiodid (DAPI/PI).
As material were used chromosome prerpations at least 24 hours after pretreatment
or slides used previously for FISH.
Procedure
- prepare new staining solution and store protected form light
- incubate chromosome preparations in 60 ml staining solution in a dark place at least overnight at room temperature
- rinse slides in PBS and remove access of liquid
- cover chromosome preparations in 10 µl of mounting medium and leave in the dark for at least 30 min before examination in fluorescence microscope under UV
Chemicals and solutions
- DABCO: Diazabicyclo[2.2.2]octan (Sigma)
- DAPI stock solution: 0.2 mg/ ml
- 4',6-Diamidin-2-phenyl-indol2HClH20 (Serva) in Aqua dest.
- Propidiumiodid stock solution: 1 mg/ml Propidiumiodid in Aqua dest.
- staining solution: 20 µl DAPI stock solution and 20 µl PI stock solution in 60 ml PBS
- PBS: 0.13 M NaCl, 0.007 M NaH2PO4, 0,03 M Na2HPO4, pH 7.4
- Mounting medium, either: a) Vectashield (Serva) or b) 1 part Tris-HCl pH 7.4
and 9 parts Glycerin with 2.3% (w/v) DABCO and 0.02% NaN3
- DABCO: Diazabicyclo[2.2.2]octan (Sigma)
- DAPI stock solution: 0.2 mg/ ml
- 4',6-Diamidin-2-phenyl-indol2HClH20 (Serva) in Aqua dest.
- Propidiumiodid stock solution: 1 mg/ml Propidiumiodid in Aqua dest.
- staining solution: 20 µl DAPI stock solution and 20 µl PI stock solution in 60 ml PBS
- PBS: 0.13 M NaCl, 0.007 M NaH2PO4, 0,03 M Na2HPO4, pH 7.4
- Mounting medium, either: a) Vectashield (Serva) or b) 1 part Tris-HCl pH 7.4 and 9 parts Glycerin with 2.3% (w/v) DABCO and 0.02% NaN3