Combining In Situ Hybridization with Retrograde Tracing and Immunohistochemistry for Phenotypic Characterization of Individual Neurons - Drugs of Abuse: Neurological Reviews and Protocols - page 136

3. Resuspend the pellet in 50 µ L of 1X TE for oligonucleotides and cDNAs and 24

µ L of 1X TE- 50 m M DTT for cRNA probes.

4. For oligonucleotide and cDNA probes, dilute the probes into 35-50 µ L of the

appropriate hybridization buffer to a concentration of 30 pg/ µ L (for cDNA

probes, approx 150,000 cpm/slide) or 3 pg/ L (for oligonucleotide probes,

approx 100,000-300,000 cpm/slide), and hybridize overnight at 40°C in a
humidifi ed chamber.

µ

5. For cRNA probes, dilute the probe into 50 L of the appropriate hybridization

buffer to a concentration of 1-5 ng/slide (0.5

µ

10 6 - 2 10 6 cpm/slide), and

× ×

hybridize overnight at 55°C in a humidifi ed chamber.

3.3.3. Post-Hybridization Washes for cDNA and Oligonucleotide Probes

1. Wash the slides briefl y in cold 4X SSC.

2. Wash twice for 30 min in 1X SSC at room temperature under agitation.

3. Wash twice for 30 min in 1X SSC at 40°C under agitation.

4. For oligonucleotide probes, add an additional wash step of 30 min (twice) in
0.1X SSC at 40°C under agitation.

5. Dehydrate twice in 100% ethanol and air-dry.

3.3.4. Post-Hybridization Washes for cRNA Probes

1. Wash the slides twice 5 min in 4X SSC under agitation.

2. Wash for 15 min at 37°C in RNase A (20 µ g/mL in 0.5 M NaCl-10 m M Tris-HCl,

pH 7.0/0.25 m M EDTA).

3. Wash the slides again in 2X SSC (5 min, twice), 1X SSC (5 min), 0.5X SSC
(5 min) at room temperature, and in 0.1X SSC at 65°C (30 min, twice) under
agitation.

4. Place the slides at room temperature in 0.1 × SSC to cool.

5. Dehydrate in graded ethanol (70%, 80%, 90%, 100%) and air-dry.

3.4. Coupling In Situ Hybridization with Retrograde Tracing
Coupling in situ hybridization with retrograde tracing is usually straightfor-
ward when using fl uorescent tracers (e.g., FluoroGold) that can be visualized
under the microscope without any additional step. In such a case, there is no
interference in the modalities of detection of the different markers. Thus, as
described in the following protocol, it appears to be the easiest way to perform
coupling of the two techniques (e.g., 5 , 9 , 15 ).

1. Retrograde tracer (FluoroGold, 4% in saline) is either pressure injected or
iontophoretically injected (e.g., 8 A, 10 s on/10 s off, over 20 min according to

ref. 15 ) at the site of interest ( see Notes 2 and 3 ).

µ

2. Allow the animals to survive 5-7 d (time may vary according to specific
conditions) before perfusion with 1-4% PFA in 0.1 M phosphate buffer ( see
Note 4 ).