Hosuk:LabNotes/2014-12-4

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Human Brain Tissue : Size Test[edit]

  • Samples from Yun
    • S1: BA8 --> the same size as before (larger one) but cut half with razer
    • S2: BA22 --> smaller than BA8
  • Try at 11/19 was failed because of Pepsin(was degraded)


Procedure[edit]

  • 12/02
    1. Take tissue on a cover glass from -80C, wait for 3min. at room temp.
    2. Make 4% Formaldehyde warm at 37C for 10min
    3. Add ~1mL of 4% Formaldehyde on a tissue, incubate for 30min. at room temp.
    4. Wash with DEPC-PBS 3 times (aspirate formaldehyde --> add PBS, and aspirate --> add PBS, and aspirate --> add PBS, and aspirate)
    5. Apply PAP pen (draw circle on a cover glass) along the outside of tissue, wait for 5min.
    6. Apply cyanoacrylate adhesiveAttach (high viscosity one) to the cover glass along the edge, then attach the cover glass to the Mattek dish
      • add a little drop of DEPC-H2O on a tissue while waiting
    7. Add 0.25% Triton X-100 in DEPC-treated H2O for 15 min.
    8. Wash with DEPC-treated H2O twice
    9. Add 200 ul 0.1% pepsin in 0.1 N HCl for 15 min. at 37C (stock : 10% Pepsin in DEPC-H2O <-- 100mg in 1mL of DEPC-H2O)
    10. Wash with 2 ml PBS three times to inactivate pepsin
    11. 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
    12. Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight.


  • 12/03
    1. Skip BS(PEG)9 step, Wash with PBS once
    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 128uL
      • Buffer 20uL
      • 50mM MnCl2 10uL
      • 5M Betaine 40uL
      • CircLigaseII 2uL
    5. RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr.
    6. 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    7. Wash with 2XSSC twice, and 1X SSC twice
    8. Prepare RCA reaction mix
      • H2O 172uL
      • Buffer 20uL
      • 25mM dNTP 2uL
      • 2mM a-dUTP 4uL
      • Phi29(low conc.) 2uL
    9. Add RCA mix, incubate at 30C overnight


  • 12/04
    1. Wash with PBS once
    2. Prepare 4uL of BS(PEG)9 in 196uL PBS
    3. add 200uL BS(PEG)9 mix, incubate for 1hr at RT
    4. Wash with PBS once, add Tris pH 8.0, incubate for 30min
    5. Wash with PBS once
    6. Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide)
    7. Preheat detection Probe at 80C for 5min
    8. Add to sample and incubate at RT for 10min
    9. Wash two times for 1 minute each with 1 mL 1X PBS


Result[edit]

  • Imaging condition
    • Pinhole=2, Laser=3, PMT=620
  • Around half of tissues were gone(I'll attach pictures of remained tissues soon)
    • I think the tissues need to be permeablized enough(which make some tissues removed) in order to make rolonies in a certain level.
    • As previous results, I think 1st rolonies would not be seen if the tissues stay well …
  • There were Rolonies detected, however numbers were still small...
  • For the next try,
    • 10min Pepsin
    • Figure out which part of the brain that I'm looking at


Result Images[edit]

  • 1st Rolony Count

File:HBTest RolonyCount Table 2014-12-04.png


  • BA8

File:HBTest Rolony BA8 2014-12-04.png


  • BA22

File:HBTest Rolony BA22 2 2014-12-04.png