before loading them onto the gel. Use 1Â MOPS buffer as
running buffer. There is no need to stain the gel as ethidium
bromide is present in the 2Â RNA-loading dye.
11. We recommend to vacuum-infiltrate tissues using a vacuum
concentrator, but a general vacuum pump is also fine. In that
case apply the vacuum slowly to avoid tissue loss due to air
bubbles.
12. We have extended this step up to 5 h and never observed an
obvious difference in experimental result.
13. The tissue can be handled in the reaction tube as well, but the
transfer to a sieve makes the liquid exchange much easier and
faster. Also, any tissue sample losses during liquid exchange are
minimized.
14. The fixed/dehydrated tissues can be stored in ethanol at 4
C
for several weeks.
15. From this step onwards, the sieves can be separated and further
treated in wells of a multi-well plate, if different tissue samples
are treated.
16. From this step onwards, the sieves with samples can also be
mounted onto a liquid-handling robot system.
17. Critical Step! Proteinase K treatment can have negative effect
on tissue integrity. However, if the proteinase K concentration
is too low or if the time of proteinase K treatment is too short,
the probe may not be able to hybridize with the
protein-covered mRNA, thus resulting in a bad outcome of
the experiment. We recommend testing different proteinase K
concentrations to optimize the conditions.
18. We usually use a hybridization temperature of 65
C, which
works in most cases.
19. High probe concentration will lead to elevated background
due to unspecific binding. We usually use a series of 0.1–2%
of the original probe reaction mix in a 1 mL hybridization
mixture to identify the perfect probe concentration.
20. Which antibody you should use depends on the probe label and
the staining reaction you would like to perform. For a
DIG-labeled probe and the NBT/BCIP or Fast Red labeling
reaction, use a dilution of an anti-digoxigenin-AP-conjugate
antibody. We used a 1:2000 dilution of the listed antibody. If
you would like to perform a TSA staining reaction using the
same probe, use an anti-digoxigenin-HRP-conjugated antibody. We used a 1:1000 dilution of the listed antibody. If you
apply a fluorescein-labeled probe and a TSA staining reaction
you need an anti-fluorescein-HRP-conjugated antibody. We
used a 1:1000 dilution of the listed antibody.
RNA-FISH
19
running buffer. There is no need to stain the gel as ethidium
bromide is present in the 2Â RNA-loading dye.
11. We recommend to vacuum-infiltrate tissues using a vacuum
concentrator, but a general vacuum pump is also fine. In that
case apply the vacuum slowly to avoid tissue loss due to air
bubbles.
12. We have extended this step up to 5 h and never observed an
obvious difference in experimental result.
13. The tissue can be handled in the reaction tube as well, but the
transfer to a sieve makes the liquid exchange much easier and
faster. Also, any tissue sample losses during liquid exchange are
minimized.
14. The fixed/dehydrated tissues can be stored in ethanol at 4
C
for several weeks.
15. From this step onwards, the sieves can be separated and further
treated in wells of a multi-well plate, if different tissue samples
are treated.
16. From this step onwards, the sieves with samples can also be
mounted onto a liquid-handling robot system.
17. Critical Step! Proteinase K treatment can have negative effect
on tissue integrity. However, if the proteinase K concentration
is too low or if the time of proteinase K treatment is too short,
the probe may not be able to hybridize with the
protein-covered mRNA, thus resulting in a bad outcome of
the experiment. We recommend testing different proteinase K
concentrations to optimize the conditions.
18. We usually use a hybridization temperature of 65
C, which
works in most cases.
19. High probe concentration will lead to elevated background
due to unspecific binding. We usually use a series of 0.1–2%
of the original probe reaction mix in a 1 mL hybridization
mixture to identify the perfect probe concentration.
20. Which antibody you should use depends on the probe label and
the staining reaction you would like to perform. For a
DIG-labeled probe and the NBT/BCIP or Fast Red labeling
reaction, use a dilution of an anti-digoxigenin-AP-conjugate
antibody. We used a 1:2000 dilution of the listed antibody. If
you would like to perform a TSA staining reaction using the
same probe, use an anti-digoxigenin-HRP-conjugated antibody. We used a 1:1000 dilution of the listed antibody. If you
apply a fluorescein-labeled probe and a TSA staining reaction
you need an anti-fluorescein-HRP-conjugated antibody. We
used a 1:1000 dilution of the listed antibody.
RNA-FISH
19