spatiotemporal resolution for understanding and manipulating
nuclei in both physiological and pathological states.
Newly developed fluorescent probes with tunable photochem-
ical properties (light absorption, quantum yields, emission fluores-
cence wavelengths, emission fluorescence intensities, etc.) are
useful in live-cell imaging. When property changes are dependent
on interaction with target nucleic acids (e.g., through hybridiza-
tion), detecting such changes enables quick, simple, and reliable
nucleic acid detection in both fixed and live cells [11–18]. Along
the same line, we have previously applied exciton-based fluorescent
probes to detect DNA and RNA molecules in situ with multiple
color choices [19–26].
These linear, 13–50 nt oligonucleotide probes carry a single
thymine or cytosine residue labeled with a homodimer of thiazole
orange (TO, 4-[3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-
methyllidene]quinolinium iodide. The TO homodimer-thymine/
cytosine was named “D514” (doubly labeled dyes with an excita-
tion maximum at 514 nm) and the oligonucleotide probes were
named “ECHO” (exicton-controlledhybridization-sensitive fluo-
rescentoligonucleotide probes). The excitonic interaction between
the two TO dyes strongly inhibits photon release, resulting efficient
photoquenching [27, 28]. Upon hybridization, bis-intercalation of
TO into the double stranded nucleic acids both substantially
reduces the interchromophoric interaction and restricts the
energy-loss rotation around the methine bond of TO, resulting in
robust fluorescent emission from both TO dyes (seeNote 1)[29].
Taking advantage of the excellent on–off ratio of ECHO probes, we
previously developed fluorescent in situ hybridization method
(ECHO-FISH) in fixed cells, a wash-free protocol that largely
reduces the labor time associated with conventional FISH method
[19, 30, 31].
In this chapter, we describe a step-by-step protocol of ECHO-
liveFISH to label nuclear U3 snoRNA and 28rRNA in living mouse
brains. The method section is divided into four parts: (1) designing
effective sequence of ECHO probes and in vitro characterization;
(2) introducing ECHO probes to living mouse brain; (3) tissue
slice preparation for imaging; (4) typical results, control experi-
ments, and troubleshooting. Detailed protocols for ECHO probe
synthesis have been described and we encourage readers interested
in synthesizing the probes in their own lab capacity to find useful
information from those sources [21, 30, 31].2 Materials
2.1 Probes 1. ECHO-liveFISH probes targeting poly(A) RNA, 28S rRNA or
U3 snoRNA (Fig.1a, d ) (e.g., GeneDesign, Inc.)
260 Dan Ohtan Wang