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 126

Fig. 4. In situ hybridization signal obtained using an antisense and sense riboprobe
against the human preprodynorphin gene. Note the absence of specifi c hybridization
signal with the use of the sense control probe.

3. Whole hemisphere human brain cryosections can be cut even up to 100 m

thick with good hybridization results (22) . However, these large sections are not
appropriate for discrete microscopic evaluation of the hybridization signal.

µ

4. Superfrost and poly- L -lysine coated microscope slides are better than gelatin-
subbed slides for very high stringency ISHH conditions (23) . However, the
gelatin-subbed slides can work perfectly well under normal hybridization condi-
tions and is easily made in the laboratory (17) . Poly- L -lysine-coated slides can
also be prepared in the lab, but it has been more cost effective and more reliable
to buy precleaned coated slides that are immediately ready for use. Both poly-
L -lysine and Superfrost slides have proven very reliable, but Superfrost slides
are also normally available in large sizes and thus very useful for processing the
human brain sections that often require high stringency conditions (24) .

5. Slide-mounted brain sections can be stored frozen for a long period of time. We
have successfully used brain sections frozen up to even 8 yr and have obtained
good levels of hybridization signals. The hybridization signal appear to be most
susceptible to repeated freeze-thawing. Thus, slides should always be sorted in
the cold (e.g., in the cryostat or on dry ice). Storage with desiccant also helps
to prevent moisture buildup.

4.2. Brain Section Pretreatment

6. Prefi xation of the tissue is important to preserve the tissue morphology, prevent
further degradation of the RNA, and enhance penetration of the probe. The
prehybridization step is also equally critical for decreasing background signal as
other phases of the ISHH procedure. For example, improper autoclaving of the
DEPC (especially if stored in plastic bottles) and impure chloroform can lead