Daniel:Notebook/RNAFACS/2015-10-26

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RNA Scope Trial Run 5: Pretreat II (Started 10/22/2015)[edit]

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Microscopy[edit]

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[edit]

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[edit]

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).