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=20X Decoding to Compare with RNAscope= *Used DRAQ5 after to stain nuclei, cannot use sample again *[[Matt:LabNotes/2015-10-29 | BA8 DARTFISH CA12kNov14_V4 with suppv2 and fiducial beads]] **90sec pepsin incubation for minimal tissue degradation ==Finding Position to image== ===Big Plan=== *Try to match one of the same locations that [[Matt:LabNotes/2015-11-6 | Gwen took tile images with RNAscope]] **Her tile images are 5.6mm long but since this BA8 has some tissue degradation I don't need to image as much *Try to find a strip as long as possible *Use 20X objective for large FOV (584um x 584um) *Use x-step size of 500um so 84um overlap between positions can be used to stitch them together ===Reference Positions using Stage Coordinates=== *Top right of tissue: (68.87, 69.21) *Bottom right of tissue: (68.87, 53.72) *Start position to match Tile 2 of Gwen's images (66.86, 62.02) **This is 2mm from the straight edge of tissue section, approximately same as Gwen's **About 7mm from the top of tissue, approx same as Gwen's Tile 2 *End position (62.86, 62.02) **This where tissue integrity has reached a reasonable limit worth imaging **This makes total imaged distance 4.5mm *9 positions imaged = 4.5mm ==Decoding Procedure== *Used 100ul 0.5uM dcProbe1 through 5 *Cycle00 *Cycle01...Cycle05 *DRAQ5 **Added 200ul 5uM DRAQ5 (diluted in PBS from 100uM brown tube Hosuk made) to tissue and incubated 15min at RT **No wash steps, went straight to imaging ***5% 638nm laser Gain of 650 *Something odd happened to the tissue in positions 1-4 (you can see the transition in position 4) **It ruins the morphology and makes nuclei hard to distinguish **Also took images directly adjacent to positions 1-4 to substitute for nuclei counts
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