Combining In Situ Hybridization with Retrograde Tracing and Immunohistochemistry for Phenotypic Characterization of Individual Neurons - Drugs of Abuse: Neurological Reviews and Protocols - page 142

Fig. 3. Coupling in situ hybridization with immunohistochemistry. Free-fl oating
vibratome sections treated for the immunohistochemical detection of Fos proteins,
followed by in situ hybridization on slides to detect enkephalin (A) and substance P
(B) mRNAs with oligonucleotide probes in the striatum. Fos-positive nuclei appear
with dark staining and mRNAs as silver grains. The double labelings show that most
of the Fos-positive neurons (arrows) are enkephalin negative (A) but substance P
positive ( B , double arrows ) after acute amphetamine treatment. (Reprinted from Jaber
et al. Acute and chronic amphetamine treatments differently regulate neuropeptide
messenger RNA levels and Fos immunoreactivity in rat striatal neurons. Neuroscience
(1995) 65, 1041-1050 with kind permission from Dr. Mohamed Jaber and Elsevier
Science.)
2. Coupling in situ hybridization with retrograde tracing is quite straightforward
when using fl uorescent tracers because there is no technical interference in the
detection of the different markers. Indeed these fl uorescent tracers may be visual-
ized directly after fi xation of the tissue without any further immunohistochemical
steps. The protocol described in this chapter is based on the use of FluoroGold
(15) as also described by others (5 , 9) . Some authors have reported the use of other
fl uorescent tracers such as Rhodamine-latex microspheres (23) or DiL (24) .
3. When using retrograde tracers that need to be visualized after an additional step
using antibodies or silver enhancement, for example (6 , 16 , 25 , 26) , the technical
problems encountered—if there are some—will be similar to those described in
Note 5 for the coupling with immunohistochemistry.
4. Whatever the tracer, the protocol described here may be performed with cRNA,
cDNAs, or oligonucleotide probes, with a fi xation allowing good sensitivity and
appropriate preservation of the tracer. Note that a fi xation with a concentration
of 2% PFA may be a good compromise.