Retrogradely transported neuronal tracers combined with immunohistochemistry using free-floating brain sections

Immunohistochemistry has been used widely for the detection of proteins in brain tissue. The process can be performed on free-floating sections, but thicker sections are required than those required for processing on slides due to the "wear and tear" of the constant agitation that free-floating sections undergo. Immunohistochemical detection of molecules of interest such as receptors, neurotransmitters or intracellular signaling molecules is used to determine the distribution of these molecules in tissues. However, it is often of interest to simultaneously determine where the neurons under investigation may project and whether they are activated by a specific stimulus. In this chapter, we will focus on protocols that we use to combine the detection of (i) Fos-positive neurons to detect increased neuronal activity, (ii) nicotine adenine dinucleotide phosphate-diaphorase (NADPH-d) to detect nitric oxide synthase, and (iii) retrogradely transported tracers to identify specific projections.