Biomedical Engineering Reference
In-Depth Information
determine the spatial and temporal profi le of the IEGs of interest, especially in the
absence of an available antibody. In this chapter, we present an in situ hybridization
protocol with radioactive probes that has been successfully and easily used on
detecting mRNA expression level and patterns, in multiple tissue types and devel-
opmental stages including embryos (Chen et al.
2012
; Haesler et al.
2004
).
This protocol has four major steps: (I) obtaining and sectioning the brains from
behaving animals under well-controlled conditions in Sects.
9.1
-
9.2
, (II) synthesis
of radioactive RNA probes from DNA plasmids in Sects.
9.3
-
9.4
, (III) pre-
hybridization and hybridization using an oil bath and washing steps in Sects.
9.5
-
9.6
, and (IV) signal detection in Sect.
9.7
.
This method allows for handling of hundreds of slides simultaneously and
quantitative analyses of gene expression. Compared with nonradioactive probe
methods such as digoxigenin (DIG) labeling, the radioactive probe hybridization
method does not require multiple amplifi cation steps using horseradish peroxi-
dase (HRP) antibodies and/or tyramide signal amplifi cation (TSA) to detect sig-
nals of target probes. Therefore, this method provides a semi-linear relation
between signal intensity and targeted mRNA amounts for quantitative analysis.
Furthermore, compared with combination study with laser capture microdissec-
tion (LCM) and qPCR, this method allows us high-throughput mRNA expression
analysis for 100-200 sides (with 400-1,000 tissue sections) simultaneously.
Using these advantages, our group and colleagues have succeeded in discovering
neural activity correlations and novel neural structures (Burmeister et al.
2005
;
Jarvis et al.
1998
; Jarvis et al.
1997
; Mello and Clayton
1994
; Mouritsen et al.
2005
) and performing high-throughput expression analysis with hundreds of
genes (Wada et al.
2006
).
9.2
Behavioral Observation and Brain Sampling
1. Using your behavioral paradigm of interest, keep animals in a quiet state for at
least 2-3 h before performing the behavioral manipulations. Then, for at least
30 min, record the animal's behavior, such as behavioral duration, frequency,
and timing, and context (with other animals, especially opposite sex) of interest
{
Note *1
}.
2. Harvest fresh brain tissue within 5-10 min.
3. Rinse the brain tissue with 1× PBS to remove blood and feather/hair, and
care-
fully
remove away extra 1× PBS from the brain.
4. Embed the tissue into a pre-labeled plastic embedding mold [22 × 22 × 22 mm,
PolyScience, cat# 18646A-1] and fi ll with OCT tissue compound [Sakura,
cat#4583], avoiding air bubbles, and orientate the tissue as needed for
sectioning.
5. Quickly freeze the block by placing it into crushed powder of dry ice {
Note *2
}.
