RNA Detection

8. Embed multiple embryos in the cap of an Eppendorf tube (see
   Note 14) (Fig.2b). Fill the cap with OCT so that a dome of
   OCT extends from the cap.


9. Cool ~100 mL isopentane in a 250 mL beaker to
   80 Cina
   liquid nitrogen bath (seeNote 15) (Fig.2c). Use blunt-end
   tweezers to carefully immerse the sample in the precooled
   isopentane until it freezes and the OCT turns white (about
   5 s) (Fig.2d).


10. Use blunt-end tweezers to take the frozen block out of the
    isopentane. Drain off excess isopentane, wrap the block in
    plastic wrap and aluminum foil (mark stage and date on the
    foil) and store in a tightly sealed bag at
    80 C(seeNote 16).

3.2 Coating
Coverslips

1. Prepare a sonication bath with clean demi water.


2. Load selected #1.5 coverslips (seeNote 2) into a coverslip
   holder and place them in a glass container (Fig.3b).


3. Fill the container with 1:20 Mucasol in Milli-Q water until the
   coverslips are covered, and sonicate for 10 min.


4. Rinse the coverslips and the container with Milli-Q water until
   all traces of detergent are gone.


5. Place the coverslip holder back in the container and fill with
   100% ethanol. Sonicate for 10 min.


6. Drain excess ethanol from the coverslip holder. Submerge
   coverslips in 1:10 poly-L-lysine in Milli-Q water for 30 min to
   coat (seeNote 17).


7. Drain excess poly-L-lysine from the coverslip holder and let the
   coverslips air-dry overnight.

3.3 Cryosectioning 1. Set cryostat block and blade temperature to
17 C(seeNote
18 ).

2. Take a cryo-block out of the
   80 C freezer and mount it to a
   specimen stage using OCT (seeNote 19). Let the block equili-
   brate to cryostat temperature for 30 min.


3. Align the anti-roll plate parallel to the cryostat blade with a
   small distance between plate and blade to guide sections
   between.


4. Mount the sample on the cryostat so that the blade will hit the
   yolk last (seeNote 20) (Fig.3a).


5. Make 8–10μm sections and quickly mount the sections on the
   coated coverslips that you prepared under Subheading3.2 (see
   Notes 21–23) (Fig.3c).


6. Let the sections dry at room temperature for a couple of
   minutes (seeNote 23) before storing them at
   80 C(see
   Notes 24and 25 ) (Fig.3d).

150 L. Carine Stapel et al.