5 FISH on Blood or Bone Marrow Smears
91
Outline
1. 100 to 200 f.!l of uncultured EDTA-, sodium acetate-treated or heparinized blood or bone marrow aspirate is dropped on a clean dry slide,
spread over the slide surface and air dried.
2. For a conventional FISH approach without the goal of maintaining the
cellular structure of the analyzed cells/nuclei, these slides are treated
with RNase and pepsin prior to denaturation.
3. Denaturation of the slides is performed for about 5 min at 75°C. Biotinylated and/or digoxigenated probes are denatured for 5 min and
hybridized on the slides for 3 nights at 37°C in a humid chamber. After
a postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to a green signal and of the
digoxigenated probe with anti-digoxigenin-rhodamine leading to red
signals. After counterstaining of the nuclei with DAPI and addition of
antifade solution, slides can be evaluated under a fluorescence microscope.
Procedure
Blood or bone marrow smear
1. Drop 100 to 200 f.!l of blood or bone marrow (EDTA-, sodium acetatetreated or heparinized) on the one end of a dry clean slide (see comment 1).
2. Spread the fluid over the whole slide surface with the small edge of a
24x60 mm coverslip. The edge of the coverslip is dipped into the blood
or bone marrow and moved slowly - once only - over the slide and
without touching the slide surface, which could disrupt the cells.
3. Let the blood or bone marrow dry out at room temperature (= RT) for
approx. 12 h before introduction into the slide pretreatment (see below).
91
Outline
1. 100 to 200 f.!l of uncultured EDTA-, sodium acetate-treated or heparinized blood or bone marrow aspirate is dropped on a clean dry slide,
spread over the slide surface and air dried.
2. For a conventional FISH approach without the goal of maintaining the
cellular structure of the analyzed cells/nuclei, these slides are treated
with RNase and pepsin prior to denaturation.
3. Denaturation of the slides is performed for about 5 min at 75°C. Biotinylated and/or digoxigenated probes are denatured for 5 min and
hybridized on the slides for 3 nights at 37°C in a humid chamber. After
a postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to a green signal and of the
digoxigenated probe with anti-digoxigenin-rhodamine leading to red
signals. After counterstaining of the nuclei with DAPI and addition of
antifade solution, slides can be evaluated under a fluorescence microscope.
Procedure
Blood or bone marrow smear
1. Drop 100 to 200 f.!l of blood or bone marrow (EDTA-, sodium acetatetreated or heparinized) on the one end of a dry clean slide (see comment 1).
2. Spread the fluid over the whole slide surface with the small edge of a
24x60 mm coverslip. The edge of the coverslip is dipped into the blood
or bone marrow and moved slowly - once only - over the slide and
without touching the slide surface, which could disrupt the cells.
3. Let the blood or bone marrow dry out at room temperature (= RT) for
approx. 12 h before introduction into the slide pretreatment (see below).