Stain structures for pSer129 and scan with high-resolution (pixel size of 6.25µm).

Import the images into ImageJ.

Convert to grayscale.

Adjust signal intensity to a standardized threshold of 95%. (All thresholds, exposure settings, and laser intensities were applied consistently across all scans).

Employ the "Process - Binary - Convert to Mask" function.

The mask function can be determined using the default method against a black background.

Apply a watershed filter to select clusters with similar distribution.

Execute the "Analyze Particles" function.

Size range: 0 to infinity

Display and summarize results.

This will extract all clusters including their coordinates and areas.

To extract clusters with a size equal to or smaller than 1 pixel (6.25µm²) and clusters identified as artifacts (>15000 µm²), use the Excel COUNTIF function, count the number of small clusters (6.25 to 50 pixels), medium-sized clusters (50 to 200 pixels), and large clusters (>200 pixels).

Determine relative density of clusters for the amount of tissue area analyzed.