Retrograde Labeling of S1 Cortex in Mice

Mice were deeply anesthetized with isoflurane, placed in a heated stereotactic frame, and injected subcutaneously with local anesthetic (bupivacaine) and analgesic (meloxicam). The skull over the right primary somatosensory (S1, barrel) cortex was exposed and a flap of bone (approx. 2×3 mm) outlined with a ¼ mm drill was removed together with the dura mater. A filter paper circle, pre-saturated with Fast Blue dye solution (FB, Polysciences, Warrington, PA, USA; 1% in distilled water) and allowed to dry, was cut to size, dipped in cortex buffer (composition: 125 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM CaCl₂, 2 mM MgSO₄), placed over the exposed pial surface and covered with Kwik-Cast silicon sealant (World Precision Instruments). The skin incision was then closed, and mice were allowed to recover. Criteria for successful retrograde labeling are indicated below.