Daniel:Notebook/HiResChrPaint/2014-8-6
No Wash FISH[edit]
Microscopy[edit]
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[edit]
- 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[edit]
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. Most photos shown here do show positive samples, although plenty of cells without signal can be seen also. From the Beliveau protocol I read that the success should be closer to 90%.
Sample B[edit]
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[edit]
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.
- 20140806 DMD-488 Dilute PreWash.lif DMD488 D1 ch00.jpg
Cells stained with DMD, Alexa 488 (green), field 1
- 20140806 DMD-488 Dilute PreWash.lif DMD488 D2 ch00.jpg
Cells stained with DMD, Alexa 488 (green), field 2
There is not strong signal shown in the majority of cells. This probably makes sense, however, since the concentration of probe is considerably less than the other samples.
Post taking these images, I returned the samples to the incubator and set the incubator to 37 C, not 42 like I usually do. They will hybridize more overnight, and I will take more images tomorrow.