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