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
- Take tissue on a cover glass from -80C, wait for 3min. at room temp.
- Make 4% Formaldehyde warm at 37C for 10min
- Add ~1mL of 4% Formaldehyde on a tissue, incubate for 30min. at room temp.
- Wash with DEPC-PBS 3 times (aspirate formaldehyde --> add PBS, and aspirate --> add PBS, and aspirate --> add PBS, and aspirate)
- Apply PAP pen (draw circle on a cover glass) along the outside of tissue, wait for 5min.
- 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
- Add 0.25% Triton X-100 in DEPC-treated H2O for 15 min.
- Wash with DEPC-treated H2O twice
- 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)
- Wash with 2 ml PBS three times to inactivate pepsin
- 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
- Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight.
- 12/03
- Skip BS(PEG)9 step, Wash with PBS once
- 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
- Wash with nucleaus-free H2O twice
- Add CircLigase II mix, incubate at 60C for 2hr.
- H2O 128uL
- Buffer 20uL
- 50mM MnCl2 10uL
- 5M Betaine 40uL
- CircLigaseII 2uL
- 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
- Wash with 2XSSC twice, and 1X SSC twice
- Prepare RCA reaction mix
- H2O 172uL
- Buffer 20uL
- 25mM dNTP 2uL
- 2mM a-dUTP 4uL
- Phi29(low conc.) 2uL
- Add RCA mix, incubate at 30C overnight
- 12/04
- Wash with PBS once
- Prepare 4uL of BS(PEG)9 in 196uL PBS
- add 200uL BS(PEG)9 mix, incubate for 1hr at RT
- Wash with PBS once, add Tris pH 8.0, incubate for 30min
- Wash with PBS once
- Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide)
- Preheat detection Probe at 80C for 5min
- Add to sample and incubate at RT for 10min
- 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