Matt:LabNotes/2015-11-11
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20X Decoding to Compare with RNAscope[edit]
- Used DRAQ5 after to stain nuclei, cannot use sample again
- BA8 DARTFISH CA12kNov14_V4 with suppv2 and fiducial beads
- 90sec pepsin incubation for minimal tissue degradation
Finding Position to image[edit]
Big Plan[edit]
- Try to match one of the same locations that 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[edit]
- 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[edit]
- 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