si:tagging:tagging2014mousestain

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 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 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