Fig. 3: Multiplex senescence marker detection with iCLAP-IF reveals subpopulations of cells with distinct marker expression patterns in a human pancreatic tissue.

a Schematic of the 6-plex iCLAP-IF workflow for senescence marker detection. P16, P53, and P21 were stained and imaged in the first round, while 53BP1, HMGB1, and Lamin B1 were detected in the second round. TSA was used for P16, P53, P21, and 53BP1 detection, whereas HMGB1 and Lamin B1 were visualized using conventional immunofluorescence. b Whole slide image of a healthy pancreatic tissue section from a 63-year-old donor stained with 6-plex senescence markers. c Zoom-in fluorescence and H&E images of a representative islet of the Langerhans region. The observed patterns were reproduced in 5 adjacent independent repeats within the same tissue sample (SENPAN_2) and across three independent human pancreatic tissue samples (SENPAN_1, SENPAN_2, and SENPAN_4; see Supplementary Data 1). d A heatmap displaying K-means clustering results based on stained senescence marker expression in over 70,000 cells of the pancreatic parenchyma selected from ten non-overlapping ROIs (2000 × 2000 px; ~1.3 × 1.3 mm each). e A heatmap shows the intensity profiles of 9 clusters. The bar graph represents the occurrence of cells in each cluster. f UMAP analysis of cell senescence marker expression. The colors highlight different clusters. g Cells in cluster 9 are predominantly located in the Islets of Langerhans. H&E images are from an adjacent section of the iCLAP-IF-stained tissue.