Vital Stain to Study Cell Invasion in Modified Boyden Chamber Assay

24–48 h, and the solution was then replaced with 0.5 M EDTA, 0.5% paraformaldehyde, or 1% paraformaldehyde, each in CMF-PBS. Vials were stored at 4°C at all times. We can report that 11 months storage in 0.5 M ETDA, and 19 months in 0.5% and 1% paraformaldehyde, both soft-tissue and skeletal metastases still fluoresce (Figure 2, A and B). Under these conditions, a few metastases lost considerable fluorescence and were only slightly visible above background. By contrast, the majority of samples stored concurrently in 4% paraformaldehyde no longer fluoresced. Background auto-fluorescence commonly increases following fixation, and the intensity of GFP fluorescence is sometimes reduced compared to fresh tissue (Figure 2, C and D). Nonetheless, maintenance of fluorescence, along with relatively good preservation of cell morphology when tissues are routinely sectioned, renders this inconvenience acceptable for most uses (Figure 2, E–G). This technique now provides investigators adequate time to thoroughly examine tissues in largescale experiments involving several replicates in multiple experimental groups. The safety of 0.5 M EDTA for both decalcification and storage also eliminates the need for additional solution changes and enables immediate histological processing of archived samples, including use for standard histological staining. In conclusion, 0.5 M EDTA in CMFPBS is capable of decalcifying murine bones in at least 24 h without harming GFP fluorescence and can be utilized for long-term archival of fluorescent specimens. Additionally, extended storage in 0.5%–1% paraformaldehyde maintains tissue fluorescence without concomitant decalcification.