To begin, obtain Isolectin B4 or IB4 and DAPI-stained mouse lung tissue sections. Acquire images under a fluorescence microscope with a 10x objective. Import the images to ImageJ software and select them.
Split the channels accordingly and load the file in ND2 format for DAPI and IB4 channels. Navigate to Analyze, Set Scale. Configure the image parameters, and then select Global, followed by OK.To set the same display range, go to Image, adjust brightness contrast, and click Set.
Set display range for IB4 as zero to 10, 000, and that for DAPI as zero to 20, 000. To eliminate the background, click Process, followed by math. Subtract the background signal based on the values detected by the magic wand tool on the background.
For selecting the best threshold value, go to Image and click Duplicate. Select both Original and Duplicate Image type to 8-bit. Next, under the Image tab, select Adjust followed by Threshold and Enter Modes.
After choosing the most realistic threshold, select Dark Background and apply. Using the magic wand tool, select larger blood vessels compared to the duplicated image, then click Delete to eliminate IB4-positive signals from large arteries and veins. To count the number of target signals, click Analyze, followed by Set Measurements, and tick Area, Limit to threshold and Display label.
Then select Analyze Particles and modify Size as zero to infinity, Circularity as zero to one, and Show as Overlay Masks. Tick Summarize, and click OK.Finally, choose Summary. Click File and save the results.
The percentage of the IB4-positive foci in the cranial, middle, caudal, accessory, and left lung lobes could be distinguished between the young and middle-aged mice using ImageJ analysis.
