156
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
Bone marrow
smears
I Subprotocol 6
Bone Marrow Smears
Successful in situ hyhridization to hone marrow smears was described by
several groups (Lee et al. 1993; Anastasi et al. 1991; Bentz et al. 1993a). The
following protocol is suitable for hone marrow smears stored for several
months (Lee et al. 1993). Depending on the probe used for hyhridization,
a proteolytic digestion step may he needed to obtain sufficient signal intensities. However, this may significantlyimpair the morphological evaluation
of the samples.
Materials
• Digestion solution: 1 J..tg/ml proteinase K dissolved in phosphate-buffered saline (PBS) (see note below).
• Phosphate-bufferedsaline (PBS).
• Methanol/acetic acid (3:1).
• 70% acetic acid.
• RNase stock solution: 10 mg/ml RNase dissolved in water. In order to
inactivate DNase, heat at 100°C for 15 min, and keep at room temperature for > 15 min.
• Hybridization mix: 50% formamide, 2x SSC, 10% dextran sulfate.
• 70%, 90%, 100% ethanol.
Procerlure
1. Wash Wright's-stained bone marrow smears for 5 min in PBS.
2. Incubate in digestion solution for 7-8 min at room temperature (see note
below).
3. W ash 3 tim es in PBS, then 3 times in water.
4. Incubate in methanol/acetic acid (3:1) for 45 sec at room temperature
(first fixation step ).
5. Incuhate in 70% acetic acid for 40 sec at room temperature (second fixation step).
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
Bone marrow
smears
I Subprotocol 6
Bone Marrow Smears
Successful in situ hyhridization to hone marrow smears was described by
several groups (Lee et al. 1993; Anastasi et al. 1991; Bentz et al. 1993a). The
following protocol is suitable for hone marrow smears stored for several
months (Lee et al. 1993). Depending on the probe used for hyhridization,
a proteolytic digestion step may he needed to obtain sufficient signal intensities. However, this may significantlyimpair the morphological evaluation
of the samples.
Materials
• Digestion solution: 1 J..tg/ml proteinase K dissolved in phosphate-buffered saline (PBS) (see note below).
• Phosphate-bufferedsaline (PBS).
• Methanol/acetic acid (3:1).
• 70% acetic acid.
• RNase stock solution: 10 mg/ml RNase dissolved in water. In order to
inactivate DNase, heat at 100°C for 15 min, and keep at room temperature for > 15 min.
• Hybridization mix: 50% formamide, 2x SSC, 10% dextran sulfate.
• 70%, 90%, 100% ethanol.
Procerlure
1. Wash Wright's-stained bone marrow smears for 5 min in PBS.
2. Incubate in digestion solution for 7-8 min at room temperature (see note
below).
3. W ash 3 tim es in PBS, then 3 times in water.
4. Incubate in methanol/acetic acid (3:1) for 45 sec at room temperature
(first fixation step ).
5. Incuhate in 70% acetic acid for 40 sec at room temperature (second fixation step).