RNA Detection

2. Prehybridization: After rinsing the fixative on three drops of
   PBS at 37C, incubate the grids (sections side down) in pre-
   hybridization buffer for 15 min at 37C, followed by 5 min on
   a hot plate at 55C(seeNote 7).


3. Denature the probe at the final concentration of 2μg/mL of
   hybridization buffer in a boiling water for 10 min followed by
   cooling down on ice for 2 min (seeNote 8).


4. Hybridization: This is performed at at 55C (forgrkmRNA,
   seeNote 7) in a formamide-saturated humid chamber made a
   6 cm petri dish covering a piece of Parafilm that is placed on top
   of filter papers soaked in 50% formamide.


5. Place cutoff caps of 0.5 mL microfuge tubes flat-side down on
   the Parafilm and fill them with 200μL hybridization buffer
   containing the denatured probe.


6. Place the chamber on a hot plate at 55C and transfer the grids
   onto the probe-containing hybridization buffer in the micro-
   fuge caps. Cover the caps with 6 cm petri dish, cover again with
   a 9 cm petri dish (to avoid evaporation) and incubate for
   overnight. It is important to ensure that the chamber will be
   moist for 16 h. Excess filter papers and formamide can be used
   (seeNote 5) (Fig.2)[5].

3.4 Double
Immunolabeling (IEM)
of mRNA and Protein

1. After rinsing the grids in prehybridization buffer and cooling
   down at RT, float the grids section side on PBS, then on
   0.15 w/v % glycine in PBS (glycine reduces the residual alde-
   hyde groups from the fixative to enable antibody reactivity) and
   then on 1% BSA in PBS to reduce nonspecific binding of the
   antibodies during the next steps.


2. Detect the antisense-tagged probe using a primary antibody
   to the tag (DIG and biotin) and PAG conjugates as described
   [4, 8].


3. Incubate the grid with anti-biotin or anti-DIG antibody:
   (a) For biotinylated probes: Incubate grid for 1 h on a 5μL
   droplet of polyclonal rabbit anti-biotin antibody diluted
   in 1% BSA in PBS.
   (b) For DIG labeled probes: Incubate grid for 20 min on a
   5 μL droplet of sheep anti-DIG antibody diluted in 1%
   BSA in PBS. Then rinse in five drops of 1% BSA in PBS for
   10 min. Incubate grid for 20 min on a 5μL droplet of
   rabbit anti-sheep antibody diluted in 1% BSA in PBS.


4. Rinse in five drops of 1% BSA in PBS for 10 min.


5. Incubate grid for 20 min on a 5μL droplet of PAG (5, 10, 15,
   or 20 nm size) diluted in 1% BSA in PBS (seeNote 9).


6. Rinse in two drops of 0.1% BSA in PBS and then in PBS.


7. Stabilize the reaction by a 5 min incubation on 1% GA in PBS.

mRNA Detection by EM 183