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=RNA Scope Trial Run 5: Pretreat III= [[Daniel:Notebook/RNAFACS|Back to Calender]] ==Microscopy== ===Positive Control (20X)=== <gallery heights=300px widths=300px perrow=2 caption="Taken with 20X oil on confocal; max z-plane; Red=Draq5 (C2), Green=FEZF2 (C3); Yellow=RBFOX3/NeuN (C1)"> File:20151028_PositiveControl.lif_Series006Snapshot1_ch00.jpg|Field 1 File:20151028_PositiveControl.lif_Series007Snapshot1_ch00.jpg|Field 2 File:20151028_PositiveControl.lif_Series009Snapshot1_ch00.jpg|Field 3 File:20151028_PositiveControl.lif_Series011Snapshot1_ch00.jpg|Field 4 </gallery> It's possible that these are signal, but I can't tell because they overlap. I checked with the positive controls (POLR2A, PPIB, UBC) and they are present (according to gene cards), but I can't tell if RNA should be in the nucleus or not. Technically, most genes being transcribed probably are, but they need to be present in the nucleus for this to work. ===Experiment (20X)=== <gallery heights=300px widths=300px perrow=3 caption="Taken with 20X oil on confocal; max z-plane; Red=Draq5 (C2), Green=FEZF2 (C3); Yellow=RBFOX3/NeuN (C1)"> File:20151028_Experiment.lif_Series003Snapshot1_ch00.jpg|Field 1 File:20151028_Experiment.lif_Series005Snapshot1_ch00.jpg|Field 2 File:20151028_Experiment.lif_Series007Snapshot1_ch00.jpg|Field 3 </gallery> These, I believe, are true signal. They get close to overlap in some points, but the dots do appear distinct, so I believe this sample does probably contain signal. This means it should be visible in the 10X objective, which will be necessary if we wish to use the Cell Raft array. ===Experiment (10X)=== <gallery heights=300px widths=300px perrow=2 caption="Taken with 10X objective on confocal; max z-plane; Red=Draq5 (C2), Green=FEZF2 (C3); Yellow=RBFOX3/NeuN (C1)"> File:20151028_Experiment.lif_Series010Snapshot1_ch00.jpg|Field 1 File:20151028_Experiment.lif_Series015Snapshot1_ch00.jpg|Field 2 File:20151028_Experiment.lif_Series018Snapshot1_ch00.jpg|Field 3 File:20151028_Experiment.lif_Series021Snapshot1_ch00.jpg|Field 4 </gallery> I don't believe you can see much with these samples. I may not be using the right settings, since using the confocal the gain and laser intensity are often different depending on the objective. The fact that the 10X uses air may also be a problem. [[Category:RNAFACS]] [[Category:RNAScope]] [[Category:PretreatIII]]
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