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
- 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 (High Viscosity!!!)
- 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 15 min at 37C
- 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 12 hr.
- 06/26
- 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 148uL
- Buffer 20uL
- 50mM MnCl2 10uL
- 5M Betaine 20uL (0.5M in final reaction volume, according to Nat. Protocol paper by Jay)
- 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
- 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 172uL
- Buffer 20uL
- 25mM dNTP 2uL
- 2mM a-dUTP 4uL
- Phi29 2uL
- Add RCA mix, incubate at 30C for 18hr
- 06/27
- 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]
- 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)