Daniel:Notebook/HiResChrPaint/2014-8-23
Padlock Probes (Started 8/13/2014)[edit]
Hybridization (From 8/21/2014)[edit]
- Remove coverslip carefully and wash in pre-warmed 2X SSCT at 60C for 15 minutes
- Incubate in 2X SSCT at room temperature for 10 minutes
- Incubate in 0.2X SSC at room temperature for 10 minutes
- Dry with kimwipes; do not let the surface containing the cells touch the kimwipes
- Add 20 uL DAPI + Slow Fade Gold to center of dish
- Cover with 22x22 #1.5 coverslip; seal with nail polish
- Allow at least 30 minutes for nail polish to dry
- Slides are quite stable for several months at 4C
Microscopy[edit]
Sample A[edit]
Sample A is stained with PPRL-488 (green) and Cot1-546 (red). The padlock probes should produce strong green signal in many cells, but there is only weak signal in a very small number of cells.
- 20140823 PPRL488 Cot1546.lif Field1 ch00.jpg
Cells stained with PPRL-488 (green) and Cot1-546 (red); field 1
- 20140823 PPRL488 Cot1546.lif Field1 Zoom ch00.jpg
Cells stained with PPRL-488 (green) and Cot1-546 (red); field 1 zoom
- 20140823 PPRL488 Cot1546.lif Field2 ch00.jpg
Cells stained with PPRL-488 (green) and Cot1-546 (red); field 2
- 20140823 PPRL488 Cot1546.lif Field2 Zoom ch00.jpg
Cells stained with PPRL-488 (green) and Cot1-546 (red); field 2 zoom
- 20140823 PPRL488 Cot1546.lif Field3 ch00.jpg
Cells stained with PPRL-488 (green) and Cot1-546 (red); field 3
Sample B[edit]
Sample B is stained with Cot1-488 (green) and DMD-546 (red). The signal seems to be present in several cells, though it is difficult to determine, given the small scale of the signal (the probes target only a few kb). Zooming in to get only one or two cells helps detect the signal better. However, there does appear to be clear signal above the noise in #1 and #3. #2 is meant to show what most cells look like, containing strong Cot1 signal with very no signal from the DMD.
- 20140823 Cot1488 DMD546.lif Field1 ch00.jpg
Cells stained with Cot1-488 (green) and DMD-546 (red); field 1
- 20140823 Cot1488 DMD546.lif Field2 ch00.jpg
Cells stained with Cot1-488 (green) and DMD-546 (red); field 2
- 20140823 Cot1488 DMD546.lif Field3 ch00.jpg
Cells stained with Cot1-488 (green) and DMD-546 (red); field 3