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Table 2 ImageJ SCRIPT for .dv image (DeltaVision format) analysis

From: Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes

STEPS SCRIPT
1. Separate the different channels (C1- for DAPI-nuclear staining and C2- for TRITC-channel associated to the PLA dots) - imageName = getTitle();
- run (“Split Channels”);
- selectWindow (“C1-” + imageName);
- selectWindow (“C2-” + imageName);
- selectWindow (“C1-” + imageName);
- run (“Smooth”);
- run (“Median...”, “radius = 2”);
2. Apply threshold on C1 - setAutoThreshold (“Default dark”);
- //run (“Threshold...”);
- setOption (“BlackBackground”, false);
- run (“Convert to Mask”);
3. Apply morphological filters on C1 - selectWindow (“C1-” + imageName);
- run (“Close-”);
- run (“Open”);
4. Segment nuclei on C1 - run (“Watershed”);
- run (“Sharpen”);
- run (“Clear Results”);
5. Count individual nuclear staining on C1 - run (“Analyze Particles...”, “size = 75–550 show = [Overlay Outlines] exclude clear add”);
6. Count individual PLA dots on C2 - selectWindow (“C2-” + imageName);
- run (“Find Maxima...”, “noise = 60 output = [Single Points] exclude”);
- selectWindow (“C2-” + imageName + “Maxima”);
7. Divide by 255 on the “find maxima output” - run (“Divide...”, “value = 255”);
- run (“Set Measurements...”, “area integrated redirect = None decimal = 3”);
8. Measure the pixel values - roiManager (“Measure”);
9. Get the results of the number of PLA dots in each nucleus - String.copyResults();
- selectWindow (“C2-” + imageName + “Maxima”);
- close();