In Situ Hybridization
Protocol
Candidate gene probes were hybridized onto a panel of mouse brains from differing
developmental ages (E13, E15, P0, and P7). 300-700 bp mouse gene fragments are sub-cloned
into pBS6E vector used as a template to generate DIG-UTP (Roche) labeled transcripts
in either orientation using T3/T7 transcript kit (Riboprobe combination System,
Promega). Antisense riboprobe was generated by T3 RNA polymerase using the
template linearized by Hind 3 (Promega) and the sense-strand control was generated
by T7 RNA polymerase using the same template linearized by EcoR1.
The in situ hybridization procedure used was modified from that previously published
from the Goldowitz laboratory (e.g.,Goldowitz et al., 1990; Yang et al.,
2002). Briefly, 4% paraformaldehyde fixed mouse fetal brains were sectioned
using a cryomicrotome at –200C and kept at –800C until use.
Before hybridization, the sections were warmed to room temperature for 20 min and
then treated with 0.25% acetic anhydride in 0.1 M triethanolamine at pH 8.0 for
10 min and dehydrated in graded concentration of ethanol.
The slides are incubated with DIG-labeled cRNA probe at concentration of about 800
ng/ml in a buffer containing 50% formamide, 4X SSC (1X SSC contains 0.15 M sodium
chloride and 0.015 M sodium citrate), 1X Dehard’s solution that is consisted of
0.02% Ficoll, 0.02% BSA), 200 μg denatured salmon test is DNA, 250 μg/ml yeast
tRNA, 10% dextran sulfate in a humid chamber for 16 hrs at 550C.
After hybridization, slides are washed in 4X SSC containing 50% formamide at 550C
for 15 min, followed by one wash in 2 X SSC at the same condition without formamide.
Sections were next incubated with RNase A (1.25 μg/ml) for 30 min at 370C
to decrease non-specific labeling.
After rinsing, slides are incubated with an anti-Dig antibody (1:300) conjugated
with alkaline phosphatase (Roche) in a PBS-T buffer containing 5% NGS for 60 min
at room temperature.
After incubation, slides were washed in 0.1 M maleic buffer, pH 7.5 for three times
and then colorized using NBT/BCIP (Roche) as a substrate at concentration of 1:50
in 0.1 M Tris-HCl buffer, pH.9.5, for 2 hrs at room temperature.
After colorization, slides were post-fixed in 4% paraformaldehyde and mounted with
a liquid mounting medium (15% glycerol and 1.5% gelatin).