The MCP has adopted various tracer injection strategies in order to compile the most accurate and reliable connectivity data. Data are systematically collected in 8-week old adult male C57Bl/6 mice. Following tracer injections, the brain is sectioned along the coronal plane and immunohistologically processed for tracer visualization. Each section is 50 ┬Ám thick and one out of every four sections across the entire brain is presented in a single series in the iConnectome viewer. All sections are counterstained with the fluorescent Nissl stain NeuroTrace Blue to provide cytoarchitectural details. The fluorescent Nissl is converted to brightfield Nissl in the image processing phase.

(1) Double coinjections. In this strategy, two injections are made into two separate delineated regions of a mouse brain. Each injection contains a mixture of an anterograde and a retrograde tracer. Phaseolus vulgaris leucoagglutinin (PHAL; anterograde) and cholera toxin subunit b (CTb; retrograde) are coinjected, while biotinylated dextran amine (BDA; anterograde) is coinjected with Fluorogold (FG; retrograde).