Combining In Situ Hybridization with Retrograde Tracing and Immunohistochemistry for Phenotypic Characterization of Individual Neurons - Drugs of Abuse: Neurological Reviews and Protocols - page 140

Fig. 2. Coupling in situ hybridization with immunohistochemistry. Free-fl oating
cryostat sections treated by in situ hybridization to detect D1 and D2 dopamine receptor
mRNAs with cRNA probes, followed by the immunodetection of calbindin (CABP)
and parvalbumin (PV) in the cortex. The primary antibodies were incubated overnight
at room temperature: monoclonal antisera to PV (1 5000 in PBS-0.3% Triton) and

CABP (1 30,000 in PBS-0.3% Triton) (from Swant., Switzerland). CABP- and PV-

positive neurons appear as dark staining and mRNAs as silver grains. Colocalization of
parvalbumin is frequently observed both with D1 receptor mRNA (A) and D2 receptor
mRNA (C) . A few CABP-immunoreactive neurons are observed, also containing D1
mRNA (B) , and almost none with D2 mRNA (D) . Arrowheads point to double-labeled
neurons, and small arrows to neurons containing only PV or CABP immunoreactiv-
ity. (Reprinted from Le Moine and Gaspar. Subpopulations of cortical GABAergic
interneurons differ by their expression of D1 and D2 dopamine receptor subtypes. Mol.
Brain Res. (1998) 58, 231-236, with kind permission of Elsevier Science.)

described (12) to detect striatal neuropeptide mRNAs to identify activated
neurons after acute or chronic amphetamine (13) .

1. Perfuse adult male rats with 250 mL of 2-4% PFA for 15 min ( see Note 9 ).

2. Dissect out the brains and post-fi x overnight in the same fi xative.