Hosuk:LabNotes/2014-12-12
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Human Brain Tissue : mild Pepsin condition test[edit]
- Samples from Yun
- S1: BA8 --> the same size as before (larger one) but cut half with Razer
- S2: BA22 --> smaller than BA8
Procedure[edit]
- 12/10
- Take Tissue glass slide from -80C, and put it in a well of sterilized 6 well plate to 50C (hot plate) for 3min. (To prevent RNase activity)
- Add 4% formamide on the glass, incubate for 30min at room temp.
- Wash tissue with 2x SSPE twice for 5min each
- Aspirate 2x SSPE, and wipe solution around the edge of the glass
- Apply adhesive to glass, place glass to MatTek dish, wait for 20min for complete dry
- Wash with 2x SSPE twice (add DEPC-PBS on a well, aspirate)
- Add 0.25% Triton X-100 in 2x SSPE for 15 min
- Pepsin incubation - S2:none, S3:0.1%, S4:0.01% for 5 min each
- Wash with DEPC-H2O three times
- 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.
- 12/11
- 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
- 12/12
- 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
- 20x, 4k, Pinhole = 4, Laser = 4, PMT = 620 (580 for S4), 20x 4K
- Result : Quit good!
- I don’t know why the rolonies are quit many for this time, but it maybe SSPE? Or less pepsin!
- 0.1% Pepsin for 5 min is the best!
- Need more detail scan with 63x obj., and DAPI or DRAQ5 staining and take images.
Result Images : prelim scanning[edit]
- 20x, 4k