Fig. 2. A summary of suggested workflows when combining FLARE with DNA FISH and immunofluorescence for clearing or expansion.

(a) A basic FLARE stain consists of carbohydrate, amine, and DNA labeling and may be followed by tissue clearing for unexpanded specimens. (b) DNA FISH may be added to workflow (a) after FLARE staining, although the noncovalent DNA stain is omitted since the stain is poor after the harsh conditions used for DNA FISH due to possible dehybridization of double-stranded DNA. (c) ExM and FLARE may be combined with gelation and denaturation (or digestion) of a sample, FLARE staining, and expansion by incubation in water. (d) DNA FISH may be added to workflow (c) after FLARE staining, again omitting the nonspecific DNA stain as in (b). (e) FLARE may be combined with immunostaining and ExM by labeling of carbohydrates, labeling of amines, immunolabeling, gelation and denaturation (or digestion), labeling of DNA, and then expansion in water. In case FLARE is found to perturb downstream immunolabeling, it is possible to use alternative protocol (f). “Carb” is carbohydrate stain; “Amine” is amine stain; “DNA” is DNA stain; “Clear” is organically cleared; “Denature” is protein denaturation using sodium dodecyl sulfate; “Digest” is enzymatic protein digestion; “Expand” is expansion in water; “DNA FISH” is DNA fluorescence in situ hybridization; “Immuno” is immunolabeling with standard primary and secondary antibodies; “Immuno-biotin” is immunolabeling with biotin-labeled primary antibodies or a combination of unlabeled primary and biotin-labeled secondary antibodies.