Hosuk:LabNotes/2014-5-19
Jump to navigation
Jump to search
Rolony from Mouse Brain Tissue Section[edit]
2nd Try[edit]
- Tissues mounted on poly-d-lysine coated glass (from Yun)
- Adhesive(Cyanoacrylate adhesives, Sigma-Aldrich, Medium-viscosity, P/N : Z105899-1EA ) works very well
Procedure[edit]
- 05/17
- Attach the glass cover slip with a mounted tissue section to the Mattek dish using cyanoacrylate adhesive
- 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 20 min (S1, 10min for S2)
- Wash with 2 ml PBS three times to inactivate pepsin
- Add RT mix (Hexamer, +a-dUTP)
- Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight. Start at 3:40pm
- 05/18
- 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
- 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
- 05/19
- 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]
- S1 : 0.25% TX-100 for 15min, 0.1% Pepsin+0.1N HCL for 20min
- S2 : 0.25% TX-100 for 10min, 0.1% Pepsin+0.1N HCL for 10min
- 20x Obj.
- S1 has much more Rolonies than S2
- S2 seemed not been permiablized well
- Cell size is much smaller than PGP1F
- Length : Around 19um ~ 60um
- The length of PGP1F was about 100um ~ 150um
- Similar to NCIH1975?
- Sample 1
- Sample 2