Immunohistochemistry (IHC) is an important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment. Although there has been a considerable progress in automation and standardization of IHC, there are still many things to be considered in proper optimization and appropriate interpretation. In this review, we aim to provide possible pitfalls and useful tips for practicing pathologists and residents in pathology training. First, general procedure of IHC is summarized, followed by pitfalls and tips in each step and a summary of troubleshooting. Second, ways to an accurate interpretation of IHC are discussed, with introduction to general quantification and analysis methods. This review is not intended to provide complete information on IHC, but to be used as a basic reference for practice and publication.

Midbrain dopamine (DA) neurons are slow pacemakers that maintain extracellular DA levels. During the interspike intervals, subthreshold slow depolarization underlies autonomous pacemaking and determines its rate. However, the ion channels that determine slow depolarization are unknown. Here we show that TRPC3 and NALCN channels together form sustained inward currents responsible for the slow depolarization of nigral DA neurons. Specific TRPC3 channel blockade completely blocked DA neuron pacemaking, but the pacemaking activity in TRPC3 knock-out (KO) mice was perfectly normal, suggesting the presence of compensating ion channels. Blocking NALCN channels abolished pacemaking in both TRPC3 KO and wild-type mice. The NALCN current and mRNA and protein expression are increased in TRPC3 KO mice, indicating that NALCN compensates for TRPC3 currents. In normal conditions, TRPC3 and NALCN contribute equally to slow depolarization. Therefore, we conclude that TRPC3 and NALCN are two major leak channels that drive robust pacemaking in nigral DA neurons.

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