Histochem Cell Biol (2012) 137:11–24 subtype mRNAs in NPY mRNA-producing neurons. This protocol complements conventional double TSA-FISH protocols suited to study coexpression of high to moder- ately expressed receptor mRNA species. Additionally, a combination of double FISH and CISH provided a novel variation of triple ISH to detect coexpression of multiple mRNAs in individual neurochemically identiﬁed neural cell populations. Using these techniques, we succeeded in documenting expression of 5-HT1A and/or 5-HT2C receptors in subpopulations of NPY mRNA-producing neurons of cortical and subcortical rat telencephalic areas. The application of combined FISH/CISH presents a prac- ticable option for coexpression studies of mRNAs expres- sed at different abundance levels, has a broad applicability and can be analyzed by conventional light and ﬂuorescence microscopy. Methodical considerations To date, most double ISH methods, which have been applied for coexpression studies in the nervous system, have been limited in their applicability for different reasons. Fig. 6 Method guide for planning multiple ISH combining FISH and CISH 21 Combinations with radioactive ISH suffer from handling problems and low cellular resolution (Hara et al. 1998), non- ampliﬁed FISH methods often possess comparatively low sensitivity (Raap et al. 1995), and sometimes even TSA-FISH is not sufﬁciently sensitive as described in the present study. More recent attempts to extend the sensitivity of FISH for detection of rare mRNA species by applying further ampliﬁcation steps of TSA-FISH, for instance using an additional enzyme-labeled ﬂuorescent method, suffer from rather high unspeciﬁc staining due to the frequent formation of unspeciﬁc ﬂuorescent crystals and require continuous adaptation and control of reaction times and conditions (Breininger and Baskin 2000). The difﬁcult handling might be a reason why these ampliﬁcation meth- ods have not become widely used. A signiﬁcant advantage of the present protocol for combined TSA-FISH and CISH for simultaneous detection of multiple mRNA species is the fact that the individual ISH techniques combined are well established, and that CISH detection provides particularly high sensitivity. An additional advantage is the manage- ability of the chromogenic reaction since the rather slow development of the signal in the NBT/BCIP reaction can be 123