- (Optional) Add drug solutions directly into the soaking ACSF
when necessary. - Alternatively, the brains can be cut along the sagittal midline
into halves and “collagen-glue” them onto glass bottom dish in
an open-book configuration. Individual speckles could be
observed in both sectioned slices and also in the halved hemi-
spheres (Fig.2e, f ). - Probe delivery efficiency can be estimated after a post-fixed
DAPI staining that labels all nuclei (Fig.2g).
3.4 Typical Results,
Validation, Control
Experiments, and
Troubleshooting
3.4.1 Typical Results
- As demonstrated in Fig.3b, a large population of granule cells in
the external and inner granule layer of the cerebellum becomes
fluorescent due to RNA hybridization. Magnified images reveal
at single nuclear level foci-like fluorescence patterns similar for
U3 snoRNA and 28S rRNA that are both localized to nucleoli,
but distinct from poly (A) RNA concentrated at nuclear speckles
(Fig.3c). At this resolution, sub-nucleolar localization of U3
snoRNA and 28S rRNA cannot be distinguished (Fig. 3c).
However the two RNA species have been shown to segregate
after transcription inhibition, suggesting localization on distinct
and independent RNPs [33, 34]. - Time-lapse imaging can be performed up to hundreds of
frames within several hours due to the molecular stability of
the probes and photostability of the dyes. Movement tracking
of individual foci by monitoring changes in their position and
fluorescence intensity over time revealed that all three nuclear
RNPs are stable with little positional change detected (Fig.3f).
3.4.2 Estimating
Sensitivity and Specificity
of the liveECHO-Probes
- To understand whether the detection of focal concentration of
the target RNA is dependent on the hybridization-sensitive
property of the ECHO probes, we electroporated Cy5-d(T) 30
in parallel experiments as described in Subheading3.2. - Both probes hybridize to poly(A) tails; however, Cy5-d(T) 30
probes result in noisy background and diffuse fluorescence in
nuclei, whereas readily distinguishable nuclear speckles with
concentrated poly(A) fluorescence were observed upon elec-
troporation of D514-(U) 22. Thus, in vivo poly(A)
äFig. 2(continued) guide high-magnification imaging; (F) Bright-field (F1) and fluorescence (F2) views in a pair
of sagittally partitioned brain halves. The fluorescence is most intense in cells adjacent to the interlobular
space. Cer, cerebellum; md, midbrain. (G) Confocal images of permeabilized cerebellar slices stained with
DAPI (blue) afterin vivoelectroporation of ECHO probe (green). One out of every three cells in the affected
regions contained D514 fluorescence, indicating that delivery efficiency is roughly 30%. Images were
acquired by LSM780 (Zeiss). Scale bars: 1 mm (F) and 20μm (G). (Panels F and G are reproduced from
[24] with permission from Oxford University Press)
Nuclear RNP Dynamics in Mammalian Tissue 267