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==Microscopy== Today's microscopy looked at the GAD1/RBFOX3 pairing, which is on the other section of tissue present on the slide. First, let's just look at a nice big picture of a single field of view. ===Single Pane=== [[File:20140918_GAD1488-RBFOX3594.lif_Production_Field_ch00.jpg|center|800px|GAD1-488 (green) and RBFOX3-594 (red), single field of view]] From the overlay, we can see that there is significant amount of overlap. However, GAD1 (green) does seem to have a few spots where RBFOX3 does not show up. We would expect, I believe, an opposite result since RBFOX3 is supposed to be a fairly ubiquitous marker. Still, there is clearly nice strong signal, so this does indicate that the FISH seems to be working well for RNA. ===Tissue Sections=== <gallery widths=700px heights=700px caption="Tissue Sections, GAD1-488 (green) and RBFOX3-594 (red)"> File:20140918_GAD1488-RBFOX3594.lif_Production_HorizontalSlice_ch00.jpg|Horizontal Section File:20140918_GAD1488-RBFOX3594.lif_Production_VerticalSlice_ch00.jpg|Vertical Section </gallery> Some potentially interesting results here, or they could be spurious. In both tiles, we see that the signal drops off. What we'd hope to see is the signal drop off in one channel but stay strong/increase in the other, but unfortunately the channels seem to be mirroring one another, as above. This leads me to believe the loss of signal is microscope related. ===Tile Scans=== <gallery widths=700px heights=700px caption="Tile Scans, GAD1-488 (green) and RBFOX3-594 (red)"> File:20140918_GAD1488-RBFOX3594.lif_Production1_Tile_ch00.jpg|First tile scan File:20140918_GAD1488-RBFOX3594.lif_Production2_Tile_ch00.jpg|Second tile scan </gallery> The tile scans reveal some more startling information. The first tile scan shows great signal all throughout, but the second tile scan the signal is only present in one pane. This has to be some sort of error, which is a problem. It looks as though even in the bright field image the tissue changes dramatically in clarity from the tile where signal is present to the others. This may be due to a sudden change in elevation, or perhaps the microscope was bumped. Perhaps the light was on in the other room which bleeds into the microscope room and ruins the pictures. Maybe the lasers get tired after a certain amount of time. I'm not sure the reason, but there is a clear disadvantage to trying to use tile scans at the 20X and 63X objectives. [[Category:RNAFISH]] [[Category:Microscopy]] [[Category:20140829]]
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