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
