Supplementary Figure 5: Nucleic acid staining with propidium iodide (PI) is equivalent in formalin alone or formalin–EDC fixed cultured human embryonic kidney (HEK293) cells.

a, Low-power wide-field fluorescent photomicrographs of HEK293 cells crosslinked with formalin–EDC and stained with PI (a, left panel, red) shows positive signal in cells (n = 3 cell cultures). The nuclei were labeled (a, middle panel, Hoechst, blue) and co-localized signals (a, right panel, purple) show that PI and Hoechst penetrate the formalin–EDC fixed cell membranes. b, Representative photomicrographs of HEK293 cultured cells fixed with formalin (n = 3 cell cultures), stained with PI and Hoechst, and imaged using the same microscopy settings as in Supplementary Fig. 4a. Formalin-fixed cells showed signal for PI (b, left panel, red) and Hoechst (b, middle panel, blue). Merged images show co-localization of both stains (b, right panel). c,d, Low-power wide-field fluorescent photomicrographs of HEK293 cells crosslinked with formalin–EDC (c) or formalin alone (d), but without PI staining (negative control) show no evidence of signal nor auto-fluorescent cells (c,d, left panel). Cell nuclei were labeled (c,d, middle panel, Hoechst, blue) and merged images shown (c,d, right panel). Scale bars are (a–d) 100 µm.