Hosuk:LabNotes/2014-6-18
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Rolony from Mouse Brain Tissue Section : 7th Try[edit]
- Tisses that I've got were not fixed
- I've discussed with Yun at 6/11, and I need to fix tissue with 4% paraformaldehyde.
- I've got 7 of 10um thickness sample and 8 of 20um thickness sample.
- And I should do careful attachment of glass cover slip on MatTek dish, otherwise cover slip is easily fell off.
- This 7th try, I test 10um and 20um sample.
Procedure[edit]
- 06/16
- Put DEPC-PBS in 3 wells of 6-well plate, and wash fixed tissue y deeping in series of PBS
- Dry PBS from cover slip and attach the glass cover slip with a mounted tissue section to the MatTek dish using cyanoacrylate adhesive (More carefully!!!))
- Wash twice using DEPC-treated H2O for 5 min each
- Add 0.25% Triton X-100 in DEPC-treated H2O for 15 min (S1, 10min for S2) and aspirate
- Wash with DEPC-treated H2O twice
- Add 200 ul 0.1% pepsin in 0.1 N HCl for 10 min at 37C
- Wash with 2 ml PBS three times to inactivate pepsin
- Add RT mix (Hexamer, -a-dUTP)
- DEPC-H2O 159uL
- M-MuLV Buffer 20uL
- 25mM dNTP 4uL
- 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.
- 06/17
- Wash with PBS once, skip BS(PEG)9
- 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
- Aspirate and add 2XSSC, incubate at 60C for 10min
- Aspirate and Wash with 2X SSC, 1X SSC and PBS once each
- Prepare RCA reaction mix
- H2O 168uL
- Buffer 20uL
- 25mM dNTP 4uL
- 2mM a-dUTP 4uL
- Phi29 4uL
- Add RCA mix, incubate at 30C for 17.5hr
- 06/18
- 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
- Imaging
Result[edit]
- 20um covers lip was fell off after RCA Primer incubation (60C for 1hr.)
- 10um thick samples stays well on cover slip.
- Result : Tissue remains well, and there are different cell left. But not many rolonies were generated, in terms of the density of rolonies.