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==Dextran Sulfate in dcProbe Hybridization to Improve Signal Uniformity (bimodal distribution)== *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=== *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) ====Imaging Order==== #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=== *Saved images in folder 3-12-2015 *Aggregate all 40 images for each step and plot histogram ([[Media:DextranSulfateExperiment_MakeIntensityHistogram.txt | Matlab script]]) ====Log scale Y axis (intensity)==== *No significant differences [[File:DextranExp_Step1_LogYHist.JPG | 450px]][[File:DextranExp_Step2_LogYHist.JPG | 450px]]<br> [[File:DextranExp_Step3_LogYHist.JPG | 450px]][[File:DextranExp_Step4_LogYHist.JPG | 450px]]<br> ====Y axis zoomed in to show details of higher intensity counts==== *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.JPG | 450px]][[File:DextranExp_Step2_ZoomYHist.JPG | 450px]]<br> [[File:DextranExp_Step3_ZoomYHist.JPG | 450px]][[File:DextranExp_Step4_ZoomYHist.JPG | 450px]]<br> ===Ideas/Thoughts=== *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
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