Hosuk:LabNotes/2014-6-27

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Rolony from Mouse Brain Tissue Section : 9th Try[edit]

  • 8th test was failed necause of mistake (not adding RT primers)
  • Tisses that I've got were fixed with 4% Formaldehyde (by Yun)
  • High Viscosity!!!) of Cyanoacrylate adhesive (Sigma-adrich, P/N:Z105902-1EA) is perfect !!!


Procedure[edit]

  • 06/25
    1. Put DEPC-PBS in 3 wells of 6-well plate, and wash fixed tissue y deeping in series of PBS
    2. Dry PBS from cover slip and attach the glass cover slip with a mounted tissue section to the MatTek dish using cyanoacrylate adhesive (High Viscosity!!!)
    3. Wash twice using DEPC-treated H2O for 5 min each
    4. Add 0.25% Triton X-100 in DEPC-treated H2O for 15 min (S1, 10min for S2) and aspirate
    5. Wash with DEPC-treated H2O twice
    6. Add 200 ul 0.1% pepsin in 0.1 N HCl for 15 min at 37C
    7. Wash with 2 ml PBS three times to inactivate pepsin
    8. Add RT mix (Hexamer, -a-dUTP)
      • DEPC-H2O 161uL
      • M-MuLV Buffer 20uL
      • 25mM dNTP 2uL
      • 100uM random hexamer RT Primer 5uL
      • Rnase Inhibitor 2uL
      • M-MuLV reverse transcriptase 10 uL
    9. Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for 12 hr.


  • 06/26
    1. Wash with PBS once, skip BS(PEG)9
    2. Add RNase H mix, incubate for 1hr. at 37C
      • RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O
    3. Wash with nucleaus-free H2O twice
    4. Add CircLigase II mix, incubate at 60C for 2hr.
      • H2O 148uL
      • Buffer 20uL
      • 50mM MnCl2 10uL
      • 5M Betaine 20uL (0.5M in final reaction volume, according to Nat. Protocol paper by Jay)
      • CircLigaseII 2uL
    5. RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr. --> 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    6. 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    7. Aspirate and add 2XSSC, incubate at 60C for 10min
    8. Aspirate and Wash with 2X SSC, 1X SSC and PBS once each
    9. Prepare RCA reaction mix
      • H2O 172uL
      • Buffer 20uL
      • 25mM dNTP 2uL
      • 2mM a-dUTP 4uL
      • Phi29 2uL
    10. Add RCA mix, incubate at 30C for 18hr


  • 06/27
    1. Wash with PBS once, Prepare 4uL of BS(PEG)9 in 196uL PBS, add 200uL BS(PEG)9 mix, incubate for 1hr at RT
    2. Wash with PBS once, add Tris pH 8.0, incubate for 30min
    3. Wash with PBS once
    4. Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide)
    5. Preheat detection Probe at 80C for 5min
    6. Add to sample and incubate at RT for 10min
    7. Wash two times for 1 minute each with 1 mL 1X PBS
    8. Imaging


Result[edit]

  • Both samples, 10um and 20um thickness, stayed well.
  • But the rolonies were not many. 20um thickness has less rolonies, weaker signal than 10um does.
    • Probably permeablization was not enough!


File:RolonyCountTable 2014-06-27.jpg


File:Tissue 9th T10um 20um 2014-06-27.jpg


Issue of tile imaging by confocal[edit]

  • A lot of artifact along boundaries, it might be uneven brigtness.


  • Tile image (Bright field) by PMT-Trans, 10x obj. 512x512 pixel


  • Sample1

File:Confocal Tile 10xobj Sample1 06272014.jpg


  • Sample2

File:Confocal Tile 10xobj Sample2 06272014.jpg


  • Tile image of Sample 2 (Bright field) by Epi-microcope, 4x obj. (Manually moved)

File:Epi 4xobj Sample2 BF 2014-06-27.jpg