Daniel:Notebook/HiResChrPaint/2014-8-6
No Wash FISH
Microscopy
First I'll test whether the samples can be taken directly to the microscope, with the hybridization buffer still on. This will produce a higher background noise, but will hopefully still result in strong areas of signal distinguishable from the background. Since this is just a test, I'll only use the positive control (sample D, Cot1-488) for now, and I'll investigate the others pending successful trial.
Sample D
- 20140805 Cot1-488 PreWash.lif Cot1488 1 ch00.jpg
Cells stained with Cot1, Alexa 488 (green), field 1
- 20140805 Cot1-488 PreWash.lif Cot1488 2 ch00.jpg
Cells stained with Cot1, Alexa 488 (green), field 2
As expected, the background is definitely higher, but the signal is also stronger. It will be interesting to do a wash on these samples, and then examine them after. Given the success, I'm going to return the positive control to the incubator and I'll take snapshots of the other 3 samples.
Sample A
Sample A is the Dystrophin gene labeled with Alexa 488. The sample has not been washed yet, but was prepared in the same manner I normally prepare samples.
- 20140806 DMD-488 PreWash.lif DMD488 1 ch00.jpg
Cells stained with DMD, Alexa 488 (green), field 1
- 20140806 DMD-488 PreWash.lif DMD488 2 ch00.jpg
Cells stained with DMD, Alexa 488 (green), field 2
- 20140806 DMD-488 PreWash.lif DMD488 3 ch00.jpg
Cells stained with DMD, Alexa 488 (green), field 3
- 20140806 DMD-488 PreWash.lif DMD488 4 ch00.jpg
Cells stained with DMD, Alexa 488 (green), field 4
- 20140806 DMD-488 PreWash.lif DMD488 5 ch00.jpg
Cells stained with DMD, Alexa 488 (green), field 5
- 20140806 DMD-488 PreWash.lif DMD488 6 ch00.jpg
Cells stained with DMD, Alexa 488 (green), field 6
Most cells showed no signal above the high background. However, several samples did show possible hybridization. From the Beliveau protocol I read that the success should be closer to 90%.
Sample B
Sample B is the Dystrophin gene labeled with Alexa 546. The sample has not been washed yet, but was prepared in the same manner I normally prepare samples.
- Experiment002 DMD546 1 ch00.jpg
Cells stained with DMD, Alexa 546 (red), field 1
- Experiment002 DMD546 2 ch00.jpg
Cells stained with DMD, Alexa 546 (red), field 2
- Experiment002 DMD546 3 ch00.jpg
Cells stained with DMD, Alexa 546 (red), field 3
Most cells showed no signal above the high background (ex. field 3). However, several samples (field 1 and 2) did show possible hybridization. From the Beliveau protocol I read that the success should be closer to 90%.
Sample C
Sample C is the dystrophin gene labeled with Alexa 488. The sample was not vacuum centrifuged, but rather 5 uL sample was added directly to the hybridization buffer.
- Experiment002 DMD546 1 ch00.jpg
Cells stained with DMD, Alexa 546 (red), field 1
- Experiment002 DMD546 2 ch00.jpg
Cells stained with DMD, Alexa 546 (red), field 2