12 Nucleus Extraction from Formalin Fixed/paraffin Embedded Tissue
169
19. Add 15 J.lI of antifade Vectashield (CAMON Vector Laboratories
HI000), cover with coverslips and look at the results under a fluorescence microscope.
Comments
l. Nytal 55 - the nylon mesh, has to be cut in 5x 5 cm squares. Such a
square can be formed into a funnel, pinned with a stapler and used as
the required filter.
2. The resulting suspension has a certain turbidity reflecting the number
of extracted nuclei. With experience, an assessment of the number of
slides on which the suspension should be distributed, will be possible.
For the first-time user, the distribution of the suspension onto two
slides is suggested.
3. An evaluation of quantity and quality of extracted nuclei was not possible before this step of the protocol, due to crystallization of PBS salts
on the slide surface. Now they have been washed away during the steps
6-9.
4. RNase and pepsin pretreatment conditions should be tested in each
laboratory on a single slide first. Both RNase and pepsin concentrations can be too stringent, resulting in clean slides without any remaining nuclei.
5. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. If longer storage is necessary, slides are stable at -20
D C
for several months.
6. Due to the fact that DNA in archival tissues has undergone some fixation steps and has been stored up to several years, a prolonged denaturation time seems useful. Moreover, in other FISH-protocols with
denaturation times of 2-5 min only, the maintenance of available metaphase chromosomes is the main aspect, which is of no significance in
the actual protocol.
7. Incubation can be stopped - if necessary - after 48 or 96 h, as well.
While in the first case, weaker signals are possible, in the second
case some cross-hybridization problems may arise.
8. During the washing steps it is important to prevent the slide surfaces
drying out, otherwise background problems may arise.
169
19. Add 15 J.lI of antifade Vectashield (CAMON Vector Laboratories
HI000), cover with coverslips and look at the results under a fluorescence microscope.
Comments
l. Nytal 55 - the nylon mesh, has to be cut in 5x 5 cm squares. Such a
square can be formed into a funnel, pinned with a stapler and used as
the required filter.
2. The resulting suspension has a certain turbidity reflecting the number
of extracted nuclei. With experience, an assessment of the number of
slides on which the suspension should be distributed, will be possible.
For the first-time user, the distribution of the suspension onto two
slides is suggested.
3. An evaluation of quantity and quality of extracted nuclei was not possible before this step of the protocol, due to crystallization of PBS salts
on the slide surface. Now they have been washed away during the steps
6-9.
4. RNase and pepsin pretreatment conditions should be tested in each
laboratory on a single slide first. Both RNase and pepsin concentrations can be too stringent, resulting in clean slides without any remaining nuclei.
5. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. If longer storage is necessary, slides are stable at -20
D C
for several months.
6. Due to the fact that DNA in archival tissues has undergone some fixation steps and has been stored up to several years, a prolonged denaturation time seems useful. Moreover, in other FISH-protocols with
denaturation times of 2-5 min only, the maintenance of available metaphase chromosomes is the main aspect, which is of no significance in
the actual protocol.
7. Incubation can be stopped - if necessary - after 48 or 96 h, as well.
While in the first case, weaker signals are possible, in the second
case some cross-hybridization problems may arise.
8. During the washing steps it is important to prevent the slide surfaces
drying out, otherwise background problems may arise.