To preserve the intact ERK1/2 activity in vivo for fluorescence microscopy, mice were transcardially perfused before isolation of brain tissue. Thirty minutes prior to transcardiac perfusion, mice were administered with 100/10 mg/kg ketamine/xylazine. Ten minutes prior to perfusion, 20 mg/kg SNC80 (i.p.) or a corresponding volume of saline was administered to the mice. Mice were then perfused with 30 mL of cold PBS and 4% paraformaldehyde (#100503-916, VWR) and were immediately decapitated to collect the brains. The brains were fixated in 4% paraformaldehyde overnight, dehydrated in 30% sucrose, and then embedded in Frozen Section Compound (#3801480, Leica). Frozen brains were sliced at a width of 30 μm using the Leica cryostat and permeabilized in 100% methanol at - 20 °C for 10 minutes. The slices were blocked in 5 % Normal goat serum (#S26-100ml, Millipore Sigma) for an hour then stained with primary antibodies as listed in table S6. For immunofluorescence labeling, the sections were incubated in the secondary antibodies according to the previously established protocol (89 (link)) and as listed in table S6. After the final washing, the nuclei of the sections were stained and the slices were mounted on a glass slide with Vectashield® (#H-1200, Vector lab). Images were acquired with a Nikon confocal microscope and assembled in Adobe Photoshop CS6 (Adobe).