|
13. Pipet off all but 1 ml of
prehybridization buffer, then add 10 µl DIG labeled
riboprobe. Incubate overnight
at 70ºC. Prepare wash solution 1 and incubate O/N
at
70 C to allow it to come to
temperature.
NEXT
DAY:
14.
Rinse embryos with 5 ml solution 1 to remove probe. Wash
embryos 3X at 70º C
with solution 1 (30 minutes
each).
15.
Wash embryos 3X at 70º C with solution 3 (30 minutes
each).
16.
Wash embryos 3X with TBST containing 2 mM levamisole (5
minutes each).
- Heat inactivate small
quantity of chick embryo powder in 1 ml TBST by heating
to
70ºC for 30 minutes,
chill on ice.
17.
Pre-block embryos in 5 ml TBST containing 10% heat
inactivated sheep serum.
(Filter the blocking solution
through a 0.2 micron syringe filter before
using.)Leave
for 2.5 hours at room
temperature. (This prevents nonspecific binding of
antibodies.)
-Pre-incubate
0.5 ml
anti-DIG Fab-AP conjugate in 1 ml TBST containing 1%
sheep
serum and appproximately 0.3%
heat inactivated chick embryo powder (1/2000 final
dilution). Rock at 4ºC.
(To remove cross-reactivity of antibody for
embryos).
18.
Spin the pre-incubated antibody solution for 10 minutes in
microfuge. Remove
supernatent and push it
through a syringe filter . Remove pre-blocking solution
from
the embryos and add the
filtered antibody. Incubate embryos overnight at
4ºC.
NEXT
DAYS
19.
Wash embryos 3X at room temperature with TBST (5 minutes
each).
20.
Washembryos several more
times (1 hour - 30 hours each) at room temperature
with TBST.
21.
Wash 3X (10 minutes each) with CT buffer. (This puts embryos
into alkaline
phosphatase reaction
buffer).
22.
Incubate embryos 15 minutes to 2 hours with detection
solution, depending on
probe.
Keep in dark during
incubation.
23.
Leave embryos overnight in PBT at 4ºC.
24.
To enhance color, ascend and descend in MeOH/PBT series (5
minutes per wash):
25%, 50%, 75%, 100%, 100%,
75%, 50%, 25%.
Store embryos in PBT at
4ºC.
25.
(optional) Clear embryos by equilibrating in glycerol/CMFET
(1:1) followed by
glycerol/CMFET
(4:1).
|
