Biomedical Engineering Reference
In-Depth Information
purple one if (1) the global intensity of the signal is not too strong and/or (2) the
DAB signal is not amplifi ed by nickel.
12. An alternate possibility is to couple
in situ
hybridization and immunohistochem-
istry on very thin sections, allowing one to analyze the same neurons on adjacent
sections
(27-29)
. This has the advantage of minimizing technical incompat-
ibilities between the various detection procedures. Nevertheless this approach
also has several disadvantages: (1) the necessity of working on slides with very
thin sections (3-4
m semithin sections) with a lowest
sensitivity for the detection of medium to low expressed antigens or mRNAs; (2)
the diffi culty of detecting the same neurons on two adjacent sections especially
for small and medium sized neurons (except on semithin sections); (3) the
longer period of time needed to analyze the data with the necessity of using two
microscopes; and (4) for semithin sections, the problems of using inclusion into
resines (araldite or epon).
µ
m cryostat or 1-2
µ
5. Conclusions
It is worth noting that the main troubleshooting of coupling
in situ
hybridiza-
tion with other anatomical techniques is related to the compatibility of
in situ
hybridization with the immunohistochemical detection of additional markers.
Several protocols have been proposed in the present chapter to couple
in situ
hybridization with retrograde tracing and immunohistochemistry. Despite the
fact that some coupling procedures have been relatively generalized (e.g.,
in
Subheadings 3.4.
and
3.5.1.
), it is important to consider that there is no
standard protocol, especially for the coupling with immunohistochemistry.
Thus each particular coupling will have to take into account the specific
experimental conditions to optimize the two types of detections within a single
protocol. Interestingly, the development of fl uorescent
in situ
hybridization
will probably help to minimize the technical incompatibilities with immuno-
histochemistry. Today, the sensitivity of fl uorescent
in situ
hybridization for
mRNA detection in the mammalian central nervous system is still limited to
highly expressed mRNAs. Nevertheless, it can be used for neuropeptide mRNA
detection and coupling (e.g.,
ref.
23
) and will have promising applications in
the future. The progress in sensitivity and the multiplicity of detection systems
will probably also help in the wide development of triple labeling procedures,
as described by some authors
(8
,
16
,
26)
.
Acknowledgments
The author thanks Dr. Patricia Gaspar, Dr. Mohamed Jaber, and Dr. Véro-
nique Bernard, who have participated in part to the work described in the present
chapter, as well as Pr. Bertrand Bloch for his constant support. The author also
thanks Claude Vidauporte and Loïc Grattier for their help with the photographic
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