Analysis of mRNA Expression Using Double In Situ Hybridization Labeling with Isotopic and Nonisotopic Probes - Drugs of Abuse: Neurological Reviews and Protocols - page 151

12. NBT/BCIP working solution: Dilute to 1 300 in alkaline phosphatase buffer.

Prepare fresh just prior to use. Levamisole (Vector Laboratories) may be added
to a concentration of 1 m M to block peripheral-type endogenous alkaline
phosphatase.
13. Genius 6 digoxygenin oligo tailing kit (Roche).
14. Yeast tRNA.
15. Normal goat serum.
16. Alkaline phosphatase-conjugated sheep polyclonal antidigoxygenin antibodies
(Roche).
17. Proteinase K solution: 10 g/mL of Proteinase K (Sigma) in 1X PBS.

18. 70%, 80%, 95%, and 100% ethanol and xylenes.
19. DPX mounting medium (Electron Microscopy Sciences).
20. Kodak hypoclearing agent.

µ

3. Methods
In situ hybridization detection of a fi rst mRNA of interest with isotopic
probes is performed according to procedures described in Chapter 9. After this,
the same slides are used for the following in situ hybridization detection of a
second mRNA of interest with nonisotopic probes, that is, digoxigenin-labeled
probes. Digoxigenin-labeled probes are detected by using antibodies against
the digoxigenin moiety. These antibodies are usually conjugated to alkaline
phosphatase or horseradish peroxidase, either of which deposits a colored
reaction product in the presence of the appropriate substrate at the site of
the hybridization probe. The protocols here are for alkaline phosphatase-
conjugated antibodies. All steps are preformed at room temperature unless
otherwise indicated.

3.1. Pretreatment of Slides

1. Rinse slides in Kodak hypoclearing agent for 5 min ( see Note 1 ). Rinse two times
in sterile dH 2 O (15 min each) and two times in 1X PBS (15 min each).

2. Prehybridization in the detergent solution (30 min). The prehybridization
increases signal-to-noise ratio and access of probes to target mRNAs.

3. Rinse three times in 1X PBS (5 min each).

4. Incubate in the Proteinase K solution (10 min) to increase penetration of probes.
Rinse three times in 1X PBS (5 min each).

5. Dehydrate slides through graded ethanol (2 min each) and air-dry for hybridiza-
tion or store at -20°C until use.

3.2. Probe Labeling
Synthesized oligonucleotide probes for mRNAs that are frequently tested
with in situ hybridization may be commercially available. If the probe for
a particular mRNA is not available, quality oligonucleotide probes can be