Hosuk:LabNotes/2013-10-28
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Detect RAB7A, and compare to ACTB
- Use 10/22 sample (MatTek dish, S2)
- RAB7A --> ATTO550 laeled
- Resuspension --> 200uM
Preparation
- 1uL 200uM ACTB + 1uL 200uM RAB7A + 2uM Cy5 100uM 1st rolony adapter in 196 uL 2x SSC --> 2 tubes
- 1uL 200uM ACTB in 199 uL 2x SSC --> 2 tubes
- 1uL 200uM RAB7A + 199 uL 2x SSC --> 2 tubes
- 2mL 80% Formamide 2x SSC, PBS, Aspiration setup, vacuum pump
- 2x SSC, PBS
- Aspiration setup, vacuum pump
Procedure
- Strip, Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 1st round RAB7A
- Strip, imaging
- Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 2nd round ACTB
- Strip, imaging
- optional experiment
- Add 200uL (RAB7A only), image --> 1st round RAB7A only
- Strip, imaging
- Add 200uL (ACTB+1st only), image --> 1st round ACTB only
- Strip, imaging
- Add 200uL (RAB7A+1st only), image --> 2nd round RAB7A only
- Strip, imaging
- Add 200uL (ACTB+1st only), image --> 2nd round ACTB only
Confocal imaging
- 20x oil, 16bit, 2048x2048 resolution
- Pick 4 location (discontinued), Z stacking