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]
- After stripping with 80% formamide and washing with 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