Table 1 Image analysis workflow

1

‘Create project’ in QuPath

NA

2

Load all images of the experiment

NA

3

Adjust ‘Brightness & contrast’ for good visualization of the markers to the eye and apply to all images

Positive cells can be easily identified by eye

4

‘Annotate’ the region of interest in each sample

The area for analysis is delineated

5

Run ‘cell detection’ with optimized parameters for the sample

All cells are identified and delineated

6

Determine the threshold(s) of the marker of interest in ‘Detection results’

Threshold(s) for each positive cell category are established

7

Run ‘Positive cell detection’ using the optimized cell detection parameters and the threshold(s) determined in step 5

Positive cells are classified into groups depending on their signal

8

Make a script including: ‘clearDetections’ ‘selectAnnotations’ ‘runPlugin’ ◊ Positive cell detection

A script for automated image analysis

9

Run the script on the complete project using ‘Run for project’

All images are analyzed

10

‘Export measurements’ as .tsv with Export type set to Annotations

A file with the analysis data

11

Perform data and statistical analysis on the data

NA