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)

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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

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

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).