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 120

4. Dehydrate the sections in a series of escalating ethanols: 70% (1 min), 80%
(1 min), 95% (2 min), and 100% (1 min), and delipidate in chloroform for 5 min.

5. Rehydrate slightly in 100% and 95% ethanol for 1 min each, and air-dry by
standing the slides vertically in a slide holder.

The sections can be used immediately for hybridization or stored frozen
with desiccant ( see Note 8 ).

3.3. Probe Preparation
35 S-labeled riboprobes can be synthesized from (1) cDNA inserts into
transcription plasmid vectors (e.g., PGEM4Z; the transcription vector has
to be linearized prior to probe labeling with suitable restriction enzymes) or
(2) cDNA templates obtained from polymerase chain reaction (PCR) with 5

extension containing a promoter sequence (T7 and T3 are generally best). For
example, good results have been obtained from riboprobes derived from the
rat preprodynorphin synthesized from the cDNA fragment of the gene (bases
466-1101) (19) and for the human preprodynorphin, a 1.2 kb of exon 4 or a
478 fragment (bases 11-488) (20) . The following procedure is used for high
specifi c activity 35 S riboprobe labeling ( see Note 9 ).

1. Thaw the following components on ice ( see Note 10 ) and add together in a sterile
eppendorf tube at room temperature: 6 µ L of 5X transcription buffer, 3 µ L of

100 m M DTT, 5.5 µ L of 2.5 m M UTP, 1 µ L of 1 µ g/ µ L linearized plasmid

vector or PCR product, 12 µ L of [ 35 S]UTP (12.5 µ Ci/ µ L) ( see Notes 11 and 12 ),

0.6 µ L of RNase inhibitor (40 U/ µ L), 1 µ L of RNA polymerase (e.g., T3 or T7),

and 0.9 mL of DEPC H 2 O.

2. Incubate for 60 min at 37°C.

3. Add DEPC H 2 O to 50 µ L.

4. Add 0.5 µ L of RNase inhibitor (40 U/ µ L) and 1 µ L of DNase (1 U/ µ g).

5. Incubate 37°C for 10 min.

6. Bring the volume up to the capacity of the spin column with DEPC H 2 O.

7. Column purify according to the manufacturer's protocol and add 1.0 µ L of 5 M

DTT (fresh).

8. Count 1 µ L of probe on a scintillation counter.

3.4. Hybridization

1. Calculate the total amount of probe and hybridization buffer needed for the
experiment ( see Notes 13 and 14 ). The amount of buffer should relate to the area
to be covered (i.e., the coverslip area). Typically, a volume of 0.1 µ L/mm 2 is used.

Thus, for a coverslip of 24 × 40 mm, a total hybridization solution volume of

approx 96 L is place over the slide. The probe concentration generally depends

on the level of the mRNA expression and a concentration curve can be carried
out to determine the optimal probe concentration. For many moderate to highly

µ