Daniel:Notebook/RNAFACS/2015-10-26: Difference between revisions
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==Microscopy== | ==Microscopy== | ||
Today I tried microscopy again, this time using the 20X objective, and for a few images, the 10X objective. | Today I tried microscopy again, this time using the 20X objective, and for a few images, the 10X objective. My overall conclusion is that the sample may have been photobleached. I need to proceed with caution, as the [http://www.cellmicrosystems.com/ Cell Raft assay], if I choose to use it, will require the 10X objective. That being said, I believe I need to be able to see signal at 10X to use the assay correctly. I recommend doing another trial (possibly tomorrow) to test it better. | ||
===20X Objective=== | |||
<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:20151026_Experiment.lif_Series013Snapshot1_ch00.jpg|Field 1 | |||
File:20151026_Experiment.lif_Series019Snapshot1_ch00.jpg|Field 2 | |||
File:20151026_Experiment.lif_Series024Snapshot1_ch00.jpg|Field 3; This green and yellow channel overlaps, so I don't think it is true signal | |||
File:20151026_Experiment.lif_Series027Snapshot1_ch00.jpg|Field 4 | |||
File:20151026_Experiment.lif_Series030Snapshot1_ch00.jpg|Field 5 | |||
File:20151026_Experiment.lif_Series032Snapshot1_ch00.jpg|Field 6 | |||
</gallery> | |||
So overall there is some signal, maybe in some of these. I had to really crank up the gain (~800) and laser intensity (5.00%) to get signal. It may just be photobleaching. | |||
===10X Objective=== | |||
<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)"> | <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: | File:20151026_Experiment.lif_Series036Snapshot1_ch00.jpg|Field 1 | ||
File: | File:20151026_Experiment.lif_Series037Snapshot1_ch00.jpg|Field 2 | ||
File: | File:20151026_Experiment.lif_Series040Snapshot1_ch00.jpg|Field 3; threshold cranked up on green and yellow channels. No signal is present over background | ||
</gallery> | </gallery> | ||
No signal here on any channels except DRAQ5. I believe this is truly a no signal. I've cranked up the thresholding on field 3 for the probe channels to show that there is no discernible signal (from the background). | |||
[[Category:RNAScope]] [[Category:RNAFACS]] [[Category:20151021]] [[Category:Microscopy]] | [[Category:RNAScope]] [[Category:RNAFACS]] [[Category:20151021]] [[Category:Microscopy]] |
Revision as of 23:53, 26 October 2015
RNA Scope Trial Run 5: Pretreat II (Started 10/22/2015)
Microscopy
Today I tried microscopy again, this time using the 20X objective, and for a few images, the 10X objective. My overall conclusion is that the sample may have been photobleached. I need to proceed with caution, as the Cell Raft assay, if I choose to use it, will require the 10X objective. That being said, I believe I need to be able to see signal at 10X to use the assay correctly. I recommend doing another trial (possibly tomorrow) to test it better.
20X Objective
- 20151026 Experiment.lif Series013Snapshot1 ch00.jpg
Field 1
- 20151026 Experiment.lif Series019Snapshot1 ch00.jpg
Field 2
- 20151026 Experiment.lif Series024Snapshot1 ch00.jpg
Field 3; This green and yellow channel overlaps, so I don't think it is true signal
- 20151026 Experiment.lif Series027Snapshot1 ch00.jpg
Field 4
- 20151026 Experiment.lif Series030Snapshot1 ch00.jpg
Field 5
- 20151026 Experiment.lif Series032Snapshot1 ch00.jpg
Field 6
So overall there is some signal, maybe in some of these. I had to really crank up the gain (~800) and laser intensity (5.00%) to get signal. It may just be photobleaching.
10X Objective
- 20151026 Experiment.lif Series036Snapshot1 ch00.jpg
Field 1
- 20151026 Experiment.lif Series037Snapshot1 ch00.jpg
Field 2
- 20151026 Experiment.lif Series040Snapshot1 ch00.jpg
Field 3; threshold cranked up on green and yellow channels. No signal is present over background
No signal here on any channels except DRAQ5. I believe this is truly a no signal. I've cranked up the thresholding on field 3 for the probe channels to show that there is no discernible signal (from the background).