Semi-Automated Method for Analyzing Lymphatic Vessels From Immunofluorescence Images

The lymphatic vasculature (LV) plays a crucial role in regulating the trafficking of immune cells into lymph nodes during homeostasis and under inflammatory conditions. LV dysfunction has been linked to various inflammatory and neuroinflammatory diseases. Thus, assessing the LV coverage area using confocal immunofluorescence microscopy is essential for accurately determining the extent of inflammation in the tissue. However, manual selection processes may require considerable time to finalize and are inherently more prone to errors, which can compromise the consistency of results among various users and lead to imprecise measurements. Here, we have developed an improved protocol to determine the area of LV covering the intestinal and meningeal tissues. By using this protocol, regions of interest can be identified more accurately and efficiently than through manual measurement, allowing for efficient analysis of changes in LV coverage during inflammation. Colon tissue and meninges obtained from mice under different experimental conditions were stained with LV-specific antibodies. Confocal microscopy was used to capture all images. Here, the manual selection protocol was compared with our semi-automatic selection protocol using the Fiji software Threshold tool. Using this tool, we successfully automated the selection of regions of interest, achieving greater accuracy in our values and minimizing both errors and the time spent by the operator. Additionally, this tool produces much more specific selections than those performed manually, and the results are more accurate. Finally, it is essential to note that this protocol can be adapted for use with other microscopy techniques, provided the image has sufficient contrast against the background and the scale size is known. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence of Colon Tissue Section and Whole-Mount Meninges Basic Protocol 2: Semi-Automated Analysis of Lymphatic Vasculature Using the Fiji Software.