Matt:LabNotes/2015-3-12
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Dextran Sulfate in dcProbe Hybridization to Improve Signal Uniformity (bimodal distribution)[edit]
- Many FISH protocols (eg Lubeck, Cai 2014) use 10% Dextran Sulfate in hybridization buffer
- Theory is dextran sulfate excludes H2O thus increasing effective concentration of probes near the tissue
- Hope is that adding dextran sulfate will give a bimodal distribution of pixel intensities making it easy to determine a threshold for calling "OFF" states
Experiment[edit]
- Sample: BA8_S2_V4_3-4-2015
- Dye-probe: 0.5uM FISGA_Adpt (Cy3)
- 3 Conditions:
- Dye 1) 0.5uM in 30% formamide + 2X SSC (1ul 100uM dye + 20ul 20X SSC + 60ul formamide + 119ul H2O = 200ul)
- Dye 2) 0.5uM in 10% dextran sulfate + 30% formamide + 2X SSC (0.5ul 100uM dye + 10ul 20X SSC + 30ul formamide + 20ul 50% dextran sulfate + 39.5ul H2O = 100ul)
- Dye 3) 0.5uM in 10% dextran sulfate + 10% formamide + 2X SSC (0.5ul 100uM dye + 10ul 20X SSC + 10ul formamide + 20ul 50% dextran sulfate + 59.5ul H2O = 100ul)
- 3 Conditions:
Imaging Order[edit]
- Strip sample with 80% formamide
- Add 100ul Dye 1 to find 4 positions and set laser power & gain to get minimal saturated pixels while broadest distribution of intensities
- 1% laser (552nm wavelength), [V] = 590, % offset = -1.00
- Image (Named Step 1)
- Strip
- Add 100ul Dye 1 and Image (Named Step 2)
- Strip
- Add 100ul Dye 2 and Image (Named Step 3)
- Strip
- Add 100ul Dye 3 and Image (Named Step 4)
- Strip
- For all:
- preheat dyes & 80% formamide in 2X SSC to 75C
- incubate @RT 10min to hybridize and then wash with 2X SSC twice
- incubate @RT 15min to strip and then wash with 1X PBS twice
- Take 10 z-stack images for each of 4 positions (40 images for each step)
Results[edit]
- Saved images in folder 3-12-2015
- Aggregate all 40 images for each step and plot histogram ( Matlab script)
Log scale Y axis (intensity)[edit]
- No significant differences
File:DextranExp Step1 LogYHist.JPGFile:DextranExp Step2 LogYHist.JPG
File:DextranExp Step3 LogYHist.JPGFile:DextranExp Step4 LogYHist.JPG
Y axis zoomed in to show details of higher intensity counts[edit]
- No significant differences
- Step 4 (10% formamide 10% dextran sulfate) has higher intensity pixels than Step 3 (30% formamide 10% dextran sulfate)
File:DextranExp Step1 ZoomYHist.JPGFile:DextranExp Step2 ZoomYHist.JPG
File:DextranExp Step3 ZoomYHist.JPGFile:DextranExp Step4 ZoomYHist.JPG
Ideas/Thoughts[edit]
- Deconvolution could improve distribution
- Try w/ PISA mask to find threshold (distribution of PISA identified features vs distribution of background)
- Shorten RCA time to decrease max value will also decrease variance
- Try subtracting background (image after stripping or with non-matching dcProbes as background)
- Use streptavidin fluorescent beads fixed with BS(PEG)9 to use as landmarks for alignment