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*[[Hosuk:Lab_Notes|LabNote]] ===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 **Add 200uL (ACTB+1st only), image --> 1st round ACTB only '''-->done at 10/29''' **Strip, imaging **Add 200uL (RAB7A+1st only), image --> 1st round RAB7A only '''-->not done yet''' ====Confocal imaging==== *20x oil, 16bit, 2048x2048 resolution *Pick 4 location (discontinued), Z stacking ===Result=== ====RAB7A compare to ACTB==== *Add three dye probes at one time - Cy5 for 1st rolony, ATTO488 for ACTB, ATTO550 for RAB7A *RAB7A were detected more than ACTB {| {{table}} | 1st Rolonies, Cy5 ([[Media:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_1stRolony.tif|Raw]]) || | ACTB, ATTO488 ([[Media:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_ACTB.tif|Raw]]) || | RAB7A, ATTO550 ([[Media:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_RAB7A.tif|Raw]]) |- | [[File:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_1stRolony.png|450px]] || | [[File:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_ACTB.png|450px]] || | [[File:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_RAB7A.png|450px]] | |} ====Hybridizing and stripping test with imaging 4 different position==== *Add 3 dye probes (1st rolony, ACTB, and RAB7A), imaging at 4 positions *Strip, and image again *Add 3 dye probes (1st rolony, ACTB, and RAB7A), imaging at 4 positions =====Result===== *Each probes annealed again well after stripping...need to analyze more though *Montage images **[[Media:Montage1_1stRun-3Dye.png|1st Run - 3 Dyes]] **[[Media:Montage2_2ndRun-Strip.png|2nd Run - Strip]] **[[Media:Montage3_3rdRun-3Dye.png|3rd Run - 3 Dyes]]
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