Trolox

Mouse cells and tissue inside the DWC were stained with 10 µg mL⁻¹ of SAFE‐modified antibodies and incubated for ≈5 min. The staining was indistinguishable with/without blocking with 0.5% BSA‐PBS, so no blocking was used beyond initial feasibility experiments. Following staining, the tissues were washed 3× with PBS containing 100 µm Trolox (Vector labs, CB‐1000). Trolox, a water soluble vitamin E analog antioxidant, was used to minimize any C₂TCO degradation that might theoretically occur in the presence of reactive oxygen species. Following imaging, a 40 µm solution of HK‐Tz scissors was applied to the tissues. The Tz‐scissors/PBS was incubated for 20 min and then washed three times before the next round. After scission, the staining, imaging, and scission cycle was repeated for multiplexed protein profiling of the same cells. The following permanent stains were used in the last cycle: Calcein AM (Thermo Fisher, C1430) and SYTOX Red (Thermo Fisher, S34859) were used for live and dead cell staining, Hoechst 33342 (Thermo Fisher, H3570) was used for nuclear staining, and tetramethylrhodamine isothiocyanate–dextran (TMRI, MW = 500 000) was used as a vascular stain.

The following antioxidants were used in this study: Trolox (Vector Laboratories, CB-1000-2), Zeaxanthin (Santa Cruz, sc-205544), L-Sodium Pyruvate (Nacalai Tesque, 06977-34), α-tocopherol (Nacalai Tesque, 34114-54), Rutin (Nacalai Tesque, 30319-04), N-Acetyl-L-cysteine (Nacalai Tesque, 00512-84) and Ascorbic acid (Nacalai Tesque, 03420-52). Ascorbic acid (100 mM) was prepared in water and stored at −20 °C. Zeaxanthin (2 mM), α-tocopherol (10 mM), Rutin (10 mM) and NAC (100 mM) were prepared in DMSO and stored at −20 °C. Trolox (100 mM) and Sodium Pyruvate (100 mM) were stored at 4 °C. Each antioxidant was diluted in 500 µL of the culture medium at a final concentration before live cell imaging.