DiI tracing

• P4 pups were perfused intracardiacally with 4% PFA (Sigma P6148) in PBS, and the brains were dissected and kept in 4% PFA in PBS at all times after dissection. 
• Dissected P4 brains were injected with small crystals of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindo-carbocyanine perchlorate (DiI, D3911 Thermo Fisher, https://www.thermofisher.com/order/catalog/product/D3911) and 4-(4-(dihexadecylamino)styryl)-N-methylpyridiniumiodide (DiA, D3883 ThermoFisher, https://www.thermofisher.com/order/catalog/product/D3883?SID=srch-srp-D3883) using glass micropipettes. 
• Gently dry the location of injection with a Kim Wipes (Kimtech) to prevent the crystal from floating at the surface of the brain
• The crystal was put on the pipet by electrostatic forces, and gently pushed in the tissue in the location of interest
• For anterograde tracing, the dye crystals were injected unilaterally in the PrV. It was identified from the location of the cochlear nucleus, which forms a bulge laterally.
• For retrograde tracing of the PrV nuclei, the cortices were removed to expose the thalamus and DiI or DiA crystals were at the level of the VPM (lateral location, at the middle of the extend of the exposed Thalamus).
• Brains were kept at 37 ̊C for 4 weeks. Brains were cut in 80mm sections with a vibratome (Leica) and counterstained with Hoechst.
• Keep the sections at 4°c, and image them in PBS with a confocal microscope (any brand, we used Olympus FV1000). We mount the section on a slide and coverslip with PBS. Sections have to be imaged within a week of cutting.
   

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