In Situ Hybridization with Isotopic Riboprobes for Detection of Striatal Neuropeptide mRNA Expression After Dopamine Stimulant Administration - Drugs of Abuse: Neurological Reviews and Protocols - page 118

Fig. 1. Rat brain sections (sagittal orientation) processed with a riboprobe and
olionucleotide antisense probe against the preprodynorphin mRNA; same fi lm exposure
time. The preprodynorphin hybridization signals are highly abundant in the striatum
(St) and hippocampus (Hipp).
6. NBT 2 Emulsion (Kodak).
7. Ammonium acetate (Sigma).
8. Cresyl violet (Sigma).
9. Ethanol.
10. Xylene (Sigma).
11. Pertex mounting media (Histolab).
3. Methods
ISHH is typically performed with either synthetic oligonucleotide (DNA;
normally 40 bases) or ribonucleotide (RNA; normally 100-1000 bases) probes
on freshly frozen brain tissue. The experimental procedures described in the
following subheadings detail the methodology for ISHH using ribonucleotide
probes (riboprobes), as such protocols normally result in higher hybridization
signals with greater signal-to-noise ratio. Although the shorter oligonucleotide
probes should have higher tissue penetrance than the riboprobes, the resulting
hybridization signal is still much lower than with the riboprobe ( see Fig. 1 ),
most likely due to the higher amount of radiolabeled nucleotides per hybridized
mRNA molecule obtained with the riboprobes. In addition, the high stability
of the RNA-RNA (as compared to RNA-DNA and DNA-DNA) duplexes
allow for increased stringency conditions (e.g., increased temperature and low
salt concentration) to be used during the hybridization and post-hybridization
washes which result in much lower nonspecifi c background signals. Moreover,
the use of riboprobes has thus far proven better than use of oligonucleotides for
the ISHH studies of the human brain, which normally have higher background
signals (due in part to the longer postmortem intervals than animal studies).
Nevertheless, oligonucleotides are still useful, especially for highly abundant
mRNAs (15) . There are a number of ISHH riboprobe procedures that have been
published in regard to visualizing mRNA expression levels in brain sections,