10 Archival Cytogenetic Slides
143
removal can be supported by very cautious use of forceps, but nuclei can
also be lost or destroyed if this procedure is not performed cautiously
enough.
Immediately after the removal of the coverslip, the slides must be dried
at room temperature and afterwards their quality has to be controlled under the microscope to detect any unremoved embedding medium, on the
one hand, and to estimate the relation of undamaged versus damaged nuclei, on the other. If visible amounts of embedding medium are still left
behind on the slide, it has to be placed in xylene once more (however, at
room temperature now) from 20 min up to 3 days. This removal of the rest
of the embedding medium is a highly critical step and must be steadily
controlled using a phase contrast microscope. An excess of remaining embedding medium will disturb the FISH reactions, and if the xylene is left on
too long, it will destroy the interphase nuclei.
As soon as this procedure is finished, the slides are dried at room temperature for about 30 min and afterwards destained in methanol. After
another drying step, the slides can be stored now at room temperature
until they are subjected to the pre-treatment procedure.
Pre-treatment
Before the FISH procedures, the slides have to be subjected to a pretreatment which removes any traces of cytoplasm and nuclear proteins as well
as RNA, which would disturb the hybridization.
The slides are incubated for 30 min at 37°C in RNase A solution (5 III
RNase A stock + 95 III 2xSSC per slide) under a coverslip. After the removal of the latter, they are rinsed three times (4 min each) in fresh
2 x SSe. After 4 min in 1x PBS, hydrolysis removal of nuclear proteins
is performed by placing the slides at 37°C in a Coplin jar filled with
1 ml HCI and 99 ml aqua bidest (adjusted to pH 2.0), 50 III of a pepsin
solution is added. After 10 min, slides are washed for 4 min in a solution
made up of 5 ml 1 M MgCh plus 95 ml lxPBS.
Re-fixation is then performed for 10 min in 1% formaldehyde (5 mIl M
MgClz plus 95 ml 1xPBS plus 2.7 ml formaldehyde stock) and slides are
washed afterwards in a Coplin jar with 100 ml 1xPBS. Dehydration is
attained by placing the slides for 4 min in increasing strengths of ethanol
at 4°C (70% - 90% - 100%), followed by air drying.
143
removal can be supported by very cautious use of forceps, but nuclei can
also be lost or destroyed if this procedure is not performed cautiously
enough.
Immediately after the removal of the coverslip, the slides must be dried
at room temperature and afterwards their quality has to be controlled under the microscope to detect any unremoved embedding medium, on the
one hand, and to estimate the relation of undamaged versus damaged nuclei, on the other. If visible amounts of embedding medium are still left
behind on the slide, it has to be placed in xylene once more (however, at
room temperature now) from 20 min up to 3 days. This removal of the rest
of the embedding medium is a highly critical step and must be steadily
controlled using a phase contrast microscope. An excess of remaining embedding medium will disturb the FISH reactions, and if the xylene is left on
too long, it will destroy the interphase nuclei.
As soon as this procedure is finished, the slides are dried at room temperature for about 30 min and afterwards destained in methanol. After
another drying step, the slides can be stored now at room temperature
until they are subjected to the pre-treatment procedure.
Pre-treatment
Before the FISH procedures, the slides have to be subjected to a pretreatment which removes any traces of cytoplasm and nuclear proteins as well
as RNA, which would disturb the hybridization.
The slides are incubated for 30 min at 37°C in RNase A solution (5 III
RNase A stock + 95 III 2xSSC per slide) under a coverslip. After the removal of the latter, they are rinsed three times (4 min each) in fresh
2 x SSe. After 4 min in 1x PBS, hydrolysis removal of nuclear proteins
is performed by placing the slides at 37°C in a Coplin jar filled with
1 ml HCI and 99 ml aqua bidest (adjusted to pH 2.0), 50 III of a pepsin
solution is added. After 10 min, slides are washed for 4 min in a solution
made up of 5 ml 1 M MgCh plus 95 ml lxPBS.
Re-fixation is then performed for 10 min in 1% formaldehyde (5 mIl M
MgClz plus 95 ml 1xPBS plus 2.7 ml formaldehyde stock) and slides are
washed afterwards in a Coplin jar with 100 ml 1xPBS. Dehydration is
attained by placing the slides for 4 min in increasing strengths of ethanol
at 4°C (70% - 90% - 100%), followed by air drying.