RNA Detection

6. Clean the head–neck region of the anesthetized mice with a
   piece of Kimwipes wet with 70% ethanol then sterile 1PBS
   immediately before surgery and carry out the subsequent sur-
   gical procedures under a stereomicroscope.


7. Cut open the skin of the head–neck region along the midline to
   expose the underlying muscle and skull.


8. Disconnect the muscle fibers with tweezers and drill through
   the skull with a gauge 27–30 needle for injection.


9. Lower the loaded injection needle through the drilled hole into
   the interlobular space between lobule V and VI of the mouse
   cerebellum to a depth of 0.5 mm (seeNote 5);


10. Gently inject the sample solution into the interlobular space
    (seeNote 6);


11. Hand-hold a pair of tweezer-type cathode electrodes and gently
    press against the sides of the occipital region (seeNote 7).


12. Step on the foot switch to deliver electrical pulses (6 pulses of
    70 V for 50 ms-duration with 150 ms-intervals for postnatal
    day 7 mouse, Fig.2c)(seeNote 8).


13. Stitch up the cut.


14. Revive the mice by keeping them on a 37C warm plate for
    more than 3 hours to recover and return to the litter until
    further experiments.

3.3 Tissue Slice
Preparation for
Imaging

A conventional inverted confocal fluorescence microscope can be

used to observe nuclear RNP foci in individual cells given that the

working distance of the lens can reach the healthy electroporated

cells located next to the injection sites. To allow accessibility, we

slice the cerebellum into thin sections that can be mounted onto

glass bottom dishes for high-resolution single photon confocal

fluorescence imaging.

1. Quickly dissect out mouse brains and transfer into ice-cold
   artificial cerebrospinal fluid (ACSF) bubbled with a gas mixture
   of 95% O 2 /5% CO 2. Fluorescence can be observed in injected
   brain regions using a stereomicroscope (Fig.2d)


2. Embed the brains in 3% agarose/ACSF gel and section hori-
   zontal or sagittal slices (300μm-thickness) using a vibratome
   slicer.


3. Select slices under stereomicroscope and mount 2–3 slices onto
   aφ27 mm-hole glass-bottom dish, press slices down by a slice
   anchor if necessary and soak in ACSF (Fig.3a).


4. Mount the dishes on to the stage of confocal microscope
   equipped with closed chamber with 95% O 2 /5% CO 2 influx.


5. Acquire single snapshot images and time-lapse images on a
   confocal microscope (seeNote 9).

Nuclear RNP Dynamics in Mammalian Tissue 265