SYBR Gold ( λEm=537  nm ) was used as a nuclear stain,34 (link) and ATTO 655 NHS ester ( λEm=680  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-μM ATTO 655 NHS ester (Sigma-Aldrich) in 1× 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× 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  μg/ml eosin for 2 min and then rinsed in 1× PBS.