Combining In Situ Hybridization with Retrograde Tracing and Immunohistochemistry for Phenotypic Characterization of Individual Neurons - Drugs of Abuse: Neurological Reviews and Protocols - page 143

5. For coupling in situ hybridization with the immunohistochemical detection
of an antigen, several strategies theoretically can be considered. Nevertheless
these strategies have to take into account the compatibility of the experimental
conditions, and there is no standard protocol. From a general point of view,
including for combined in situ hybridization, double labeling approaches classi-
cally decrease the sensitivity of detection for both techniques. Thus the use of
cRNA probes for in situ hybridization is recommended for low mRNA expression
from an in situ hybridization aspect. However the stringency conditions that are
necessary for the use of these probes (high temperatures, presence of detergents,
RNase A treatment, etc.) may alter the histological preservation of the tissue
and consequently the quality and intensity of the immunohistochemistry. The
two methods have also to be compatible in terms of tissue fi xation. Whatever
the type of probes used, no or low fi xation gives the best sensitivity for in
situ hybridization. On the other hand, the best conditions for immunohistochemi-
cal detection depend to a large extent on the type of antigen to be detected
(neuropeptides, receptors, enzymes, etc.) but strong fixation is sometimes
required for an optimal immunohistochemical signal. Thus each experimental
procedure of coupling will have to be designed depending on the type of markers
to be visualized, and the choice for a given protocol most of the time will rely on
a “compromising” procedure allowing a satisfying visualization of the mRNA
together with the antigen.

6. When using cryostat free-fl oating sections, 4% PFA fi xation is more appropriate
to limit damaging of the 15-20- µ m sections during the processing of the various

hybridization steps

7. Cryostat free-fl oating or vibratome sections may be also stored in cryoprotectant
(30% sucrose-10% glycerol in PBS) at -80°C before use.

8. When using the peroxidase method for immunohistochemical detection, it is
recommended not to use nickel to amplify the signal obtained with DAB to avoid
further nonspecifi c radioactive hybridization signal.

m for a good cellular
resolution; thus brains need to be fixed at least with 2% PFA to allow an
homogeneous sectioning with this thickness.
10. When using cryostat sections on slides, which can be appropriate to visualize
highly expressed mRNA or antigens (e.g., for neuropeptides), the immunohisto-
chemical signal can be revealed before or after emulsion. In the fi rst case, one
needs to check for the nonspecifi c accumulation of silver grains. In the latter
case, the development of the signal throught the emulsion will decrease the
intensity of the immunohistochemical signal (20 , 21) .
11. Nonradioactive in situ hybridization using biotin- or digoxigenin-labeled
nucleotides is classically revealed by an alkaline phosphatase-conjugated
complex and gives a purple staining after incubation in nitroblue tetrazolium/5-
bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrates. Combined immu-
nohistochemistry with the peroxidase-anti-peroxidase method will give a brown
staining after incubation with DAB, which can be differentiated easily from the

9. For vibratome sections, the thickness has to be around 30 µ