Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.
Total time
~4–6 hours per tissue section (excluding antigen retrieval incubation and staining overnight steps)
Steps
1
Perform heat-induced antigen retrieval on tissue sections
Heat FFPE tissue sections using a standard protocol to expose epitopes for antibody binding. This critical preprocessing step enables subsequent immunofluorescence labeling.
▶ 01:03
2
Block nonspecific antibody binding on tissue
Apply blocking solution to FFPE sections to reduce background and nonspecific staining before immunofluorescence labeling.
▶ 01:46
3
Apply multiplexed immunofluorescence labels to tissue
Incubate tissue sections with primary and fluorescent secondary antibodies targeting immune cell markers to enable multiparameter visualization.
▶ 02:28
4
Align and register digital image layers
Use computational methods to align multichannel fluorescence images and correct spatial drift to ensure accurate colocalization analysis.
▶ 03:36
5
Detect and define tissue boundaries in images
Apply image processing algorithms to identify tumor tissue region and distinguish it from background or non-tissue areas.
▶ 04:36
6
Segment tissue into regions of analysis
Subdivide tissue area into defined regions or compartments to enable spatial quantification of immune populations.
▶ 05:35
7
Identify and quantify cells of interest
Use automated or semi-automated algorithms to detect individual immune cells matching marker criteria and enumerate populations.
▶ 06:28
8
Generate spatial heatmaps of immune cell distribution
Create visual maps displaying the spatial density and localization of immune cell populations across the tumor microenvironment.
▶ 07:15
9
Interpret representative results and imaging outputs
Review and validate representative visualizations, quantifications, and spatial maps demonstrating immune cell heterogeneity in tumor tissue.
▶ 08:10