=====Protocol for chemical labeling of mouse brain slices=====

===Vibratome slices (200 µm)===

       * fix slices in 4% PFA (in 0.1 M PB) at room temperature for 20 min in glass-well plate on nutator

       * remove fixative and and wash 2 x 5 min with PBT (0.3% Triton X-100)

       * add substrate for a final concentration of 1 µM in PBT (see [[si:kohl2014:tagging2014substrates|Notes on substrates for chemical labeling]])
       * incubate slices on nutator for 15 min //agitation of the sample(s) is important to obtain rapid, homogenous staining! long incubation times (> 6 h) can result in slightly increased background staining!//

       * wash brains with PBT (2 x 10 min) 

       * remove PBT as completely as possible and add ~300 µl of Vectashield (or other) mounting medium //we observed that subsequently exchanging this PBT-Vectashield mix for fresh Vectashield results in more homogenous signal along z//

       * mount brains and image

===Cryostat sections (30-50 µm, floating)===

      * wash 2 x 5 min with PBT (0.3% Triton X-100)

      * add substrate for a final concentration of 1 µM in PBT (see [[si:kohl2014:tagging2014substrates|Notes on substrates for chemical labeling]])

      * incubate slices on nutator for 15 min //agitation of the sample(s) is important to obtain rapid, homogenous staining! long incubation times (> 6 h) can result in slightly increased background staining!//

      * wash brains with PBT (2 x 10 min) 

      * remove PBT as completely as possible and add ~300 µl of Vectashield (or other) mounting medium //we observed that subsequently exchanging this PBT-Vectashield mix for fresh Vectashield results in more homogenous signal along z//

      * mount brains and image