Atto 655 nhs ester

SYBR Gold ( ${\lambda }_{\mathrm{Em}}=537\mathrm{nm}$ ) was used as a nuclear stain,³⁴ (link) and ATTO 655 NHS ester ( ${\lambda }_{\mathrm{Em}}=680\mathrm{nm}$ ) as a stromal/cytoplasmic stain [Fig. 2(a)]. Both of these fluorescent agents can be excited at 285 nm. In our optimized protocol, fresh tissues were stained by submerging them in a 0.825:10,000 v/v solution of SYBR Gold (Thermo Fisher, Cat. No: S11494) and $11.25\text{-}\mu \mathrm{M}$ ATTO 655 NHS ester (Sigma-Aldrich) in $1\times$ phosphate-buffered saline (PBS, Gibco, Cat. No: 10010023) at pH 8.0 for 5 min. The stained tissue was then rinsed 3 times in a large volume of $1\times$ PBS (pH 7.4) for 30 s per wash, followed by MUSE imaging.
For control experiments to compare cytoplasmic/stromal staining approaches, a published MUSE protocol with eosin staining was used.²⁹ (link) In short, tissues were stained with $200\mu \mathrm{g}/\mathrm{ml}$ eosin for 2 min and then rinsed in $1\times$ PBS.