Matt:LabNotes/2016-12-5

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Decoding 5342 Occipital Cortex with CA12kOct2016 V4[edit]

Procedure[edit]

  • Cycle00 = FISGA_Adpt Cy3 universal probe + Fiducial marker beads (also in Cy3 channel)
  • Cycle01-06 = 6 cycles of decoding
  • Stripped_After6 = After Cycle06 stripped with 2x 15min incubations of 80% formamide in 2X SSC with 2x PBS washes in between (same procedure for stripping as between every cycle)
    • Almost no signal, including Cy3, which makes me think Fiducial marker beads didn't work or else have become photobleached
  • Anti-NeuN = 488 channel containing antibody staining for neurons
  • DRAQ5 = Cy5 channel

Experiment Settings[edit]

  • Adaptive Focus Control on
    • Worked really well, did not have to change z start/end positions before each cycle
  • 1024x1024 resolution
  • Bidirectional scan on (phase ~= -32.90)
  • 8-bit
  • SIMULTANEOUS acquisition of 488 & Cy5, Cy3, & BF
    • Image order is ch00=488, ch01=Cy5, ch02=Cy3, ch03=BF
  • 10 positions of 55 z slices each (16.01um) took 29min 40sec to image all 3 channels
    • Actually 9 positions, 'Pos8' missing, and Pos9 is 110 z slices (32.02um)
    • Pos1,..,Pos7,Pos9,Pos10

Anti-NeuN Staining Procedure[edit]

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