14 Nucleus Extraction from Cryofixed Tissue
183
4. Nytal 55 - the nylon mesh has to be cut in 5 x 5 cm squares. Such a
square can be formed into a funnel, pinned by a stapler and used
as the required mter.
5. 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, a distribution of suspension on two slides is
suggested.
6. 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
17-19.
7. 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.
8. The pretreated slides can be hybridized immediately, or stored at RT
for up to 3weeks. Iflonger storage is necessary slides are stable at -20°C
for several months.
9. 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.
10. 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.
11. During the washing steps, it is important to prevent the slide surfaces
drying out, otherwise background problems may arise.
References
Dhingra, K, Sahin, A, Supak, J, Kim, SY, Hortobagyi, G, Hittelman, WN (1992) Chromosome in situ hybridization on formalin-fixed mammary tissue using non-isotopic, non-fluorescent probes: technical considerations and biological implications.
Breast Cancer Res Treat, 23:201-210
183
4. Nytal 55 - the nylon mesh has to be cut in 5 x 5 cm squares. Such a
square can be formed into a funnel, pinned by a stapler and used
as the required mter.
5. 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, a distribution of suspension on two slides is
suggested.
6. 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
17-19.
7. 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.
8. The pretreated slides can be hybridized immediately, or stored at RT
for up to 3weeks. Iflonger storage is necessary slides are stable at -20°C
for several months.
9. 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.
10. 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.
11. During the washing steps, it is important to prevent the slide surfaces
drying out, otherwise background problems may arise.
References
Dhingra, K, Sahin, A, Supak, J, Kim, SY, Hortobagyi, G, Hittelman, WN (1992) Chromosome in situ hybridization on formalin-fixed mammary tissue using non-isotopic, non-fluorescent probes: technical considerations and biological implications.
Breast Cancer Res Treat, 23:201-210