Matt:LabNotes/2017-1-13

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Sample 2 was decoded successfully but not stained with Anti-NeuN to confirm neurons

Anti-NeuN Staining Procedure[edit]

  1. Strip with 80% formamide twice and washing with 1X PBS
  2. Rinse tissue with 1X PBS for 30min
  3. Incubate section for 30min in 1ml blocking buffer
    • 50ul DKS + 250ul 10% BSA + 0.1ul TX-100 + 100ul 10X PBS + 600ul H2O
  4. Incubate section for 1hr in 1ml NeuN antibody solution
    • 1ul rabbit anti-NeuN (Millipore MABN140) diluted in 999ul blocking buffer same as in step 2
  5. Wash 3x 5min in 1X PBS
  6. Stain with Donkey anti-Rabbit Alexa 488 for 1.5hr
    • DxR488 stock was diluted 1:200 in blocking buffer without the TX-100
  7. Wash 3x 5min in 1X PBS
    • 2nd wash use 5uM DRAQ5 in 1X PBS
    • 3rd wash use 1X PBS + 0.1% Tween-20
  8. Add 150ul Vectashield mounting media with antifade and allow 10min before imaging
  9. DRAQ5 signal was very weak so: incubate in another 5uM DRAQ5 + 1X PBS for 15min and don't wash before imaging again