304
MARTIN LAWRIE, MARIA OCANA GIL, and DON CARDY
1
3
5
7
10
12
14
16
17
19
21
X
Fig. 2. Locations of first
12 spots of cell sample on
template slide. Spotting
in this manner will allow
sample to spread correctly
2. It is important that the intended sample has a sufficiently high cell
density for adequate analysis. For example, there should be at least
20 nuclei in a field of view using a x 10 objective, and there should
be at least 5 metaphase cells in each square of the template slide.
3. Pipette 2 III of methanol/acetic acid fixed cell suspension (e.g. peripheral blood lymphocytes) onto all 24 areas in a series of alternating
squares as shown below (Fig. 2). This will prevent the drops of suspension from interfering with each other as they spread.
4. Once the first group of drops has air dried, spot the remaining squares
with 2 III drops in the same manner. After the slide has dried, examination of the slide under phase contrast will reveal whether any
squares have been missed.
Hybridisation
1. Pre-warm a Chromoprobe Multiprobe Hybridisation Chamber (or a
plastic slide box measuring 130x80x30 mm) to 37°C in a water bath.
2. Mix 30 III of hybridisation solution per device by repeated pipetting
and pre-warm to 37°C. Pre-warm Multiprobe to 37°C by placing device
label side down. Avoid touching the raised boss surfaces.
3. Wash sample slide in 2x SSC for 2 min at room temperature.
4. Dehydrate sample slide through an ethanol series: 2 min each in 70%,
85% and absolute ethanol at room temperature. Air dry and place at
37°C to warm.
5. Pipette 1 III of pre-warmed hybridisation solution on to each of the
bosses on the pre-warmed device while it remains at 37°C.
6. Carefully invert the sample slide over the Multiprobe such that the
number 1, is located over the top right-hand area of the Multiprobe
(Fig. 3).
MARTIN LAWRIE, MARIA OCANA GIL, and DON CARDY
1
3
5
7
10
12
14
16
17
19
21
X
Fig. 2. Locations of first
12 spots of cell sample on
template slide. Spotting
in this manner will allow
sample to spread correctly
2. It is important that the intended sample has a sufficiently high cell
density for adequate analysis. For example, there should be at least
20 nuclei in a field of view using a x 10 objective, and there should
be at least 5 metaphase cells in each square of the template slide.
3. Pipette 2 III of methanol/acetic acid fixed cell suspension (e.g. peripheral blood lymphocytes) onto all 24 areas in a series of alternating
squares as shown below (Fig. 2). This will prevent the drops of suspension from interfering with each other as they spread.
4. Once the first group of drops has air dried, spot the remaining squares
with 2 III drops in the same manner. After the slide has dried, examination of the slide under phase contrast will reveal whether any
squares have been missed.
Hybridisation
1. Pre-warm a Chromoprobe Multiprobe Hybridisation Chamber (or a
plastic slide box measuring 130x80x30 mm) to 37°C in a water bath.
2. Mix 30 III of hybridisation solution per device by repeated pipetting
and pre-warm to 37°C. Pre-warm Multiprobe to 37°C by placing device
label side down. Avoid touching the raised boss surfaces.
3. Wash sample slide in 2x SSC for 2 min at room temperature.
4. Dehydrate sample slide through an ethanol series: 2 min each in 70%,
85% and absolute ethanol at room temperature. Air dry and place at
37°C to warm.
5. Pipette 1 III of pre-warmed hybridisation solution on to each of the
bosses on the pre-warmed device while it remains at 37°C.
6. Carefully invert the sample slide over the Multiprobe such that the
number 1, is located over the top right-hand area of the Multiprobe
(Fig. 3).