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)
  • 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

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