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

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();