448
BRIGITTE SCHLEGELBERGER et aI.
Pepsin digestion of
FISH slides
Cytospin
preparations
1. Incubate slide in a Coplin jar for 5 min at 37°C with freshly prepared
(not prewarmed!) digestion solution.
2. Wash slide once in A. bidest.
3. Fix slide for 10 min in paraformaldehyde solution (1%).
4. Wash slide once in A. bidest.
5. Dehydrate in 70%, 85% and 100% ethanol 1 min for each step.
6. Air dry slide.
For different samples, optimal pepsin concentration and incubation duration may vary slightly.
Slide preparation for cytogenetic samples, imprints and blood/bone marrow smears: Slides from cytogenetic samples are prepared as described
above. Blood and bone marrow smears as well as imprints are prepared
as for routine morphologic evaluation.
Cytospin preparations of cytogenetic specimens: The hybridization efficiency for interphase FISH from cytogenetic specimens can be enhanced
by preparing cytospin preparations. This procedure is especially recommended for samples containing few cells or displaying no signals or a high
background noise after conventional preparation.
1. Determine the cell number.
2. Add 1x PBS ad 10 ml to 1-3 ml cells containing Carnoy's fixative in a
IS-ml tube.
3. Spin 10 min at 1800 rpm in a lab centrifuge.
4. Discard supernatant.
5. Resuspend pellet in I x PBS to a cell count of I x 10
4
cells/m!.
6. Spin 200 Jll of this solution (2000 cells/slide) in a cytocentrifuge for
5 min at 800 rpm.
7. Air dry slide.
8. Proceed with pepsin digestion of FISH slides.
Cytospin preparations of cell suspensions from solid tumors: This protocol is intended for FISH analyses of frozen specimens from lymph nodes
and solid tumors, in which neither cytogenetic preparations nor freshly
prepared imprints are available.
BRIGITTE SCHLEGELBERGER et aI.
Pepsin digestion of
FISH slides
Cytospin
preparations
1. Incubate slide in a Coplin jar for 5 min at 37°C with freshly prepared
(not prewarmed!) digestion solution.
2. Wash slide once in A. bidest.
3. Fix slide for 10 min in paraformaldehyde solution (1%).
4. Wash slide once in A. bidest.
5. Dehydrate in 70%, 85% and 100% ethanol 1 min for each step.
6. Air dry slide.
For different samples, optimal pepsin concentration and incubation duration may vary slightly.
Slide preparation for cytogenetic samples, imprints and blood/bone marrow smears: Slides from cytogenetic samples are prepared as described
above. Blood and bone marrow smears as well as imprints are prepared
as for routine morphologic evaluation.
Cytospin preparations of cytogenetic specimens: The hybridization efficiency for interphase FISH from cytogenetic specimens can be enhanced
by preparing cytospin preparations. This procedure is especially recommended for samples containing few cells or displaying no signals or a high
background noise after conventional preparation.
1. Determine the cell number.
2. Add 1x PBS ad 10 ml to 1-3 ml cells containing Carnoy's fixative in a
IS-ml tube.
3. Spin 10 min at 1800 rpm in a lab centrifuge.
4. Discard supernatant.
5. Resuspend pellet in I x PBS to a cell count of I x 10
4
cells/m!.
6. Spin 200 Jll of this solution (2000 cells/slide) in a cytocentrifuge for
5 min at 800 rpm.
7. Air dry slide.
8. Proceed with pepsin digestion of FISH slides.
Cytospin preparations of cell suspensions from solid tumors: This protocol is intended for FISH analyses of frozen specimens from lymph nodes
and solid tumors, in which neither cytogenetic preparations nor freshly
prepared imprints are available.