RNA Detection

2. 2% PFA + 0.2% glutaraldehyde (GA) in 0.1 M phosphate buffer
   at pH 7.4. Prepare fresh from 16% PFA and 8% EM grade GA
   stocks.


3. 1% PFA for postfixation in 0.1 M PBS at pH 7.4 for sample
   storage. Prepare fresh from stock.


4. 1% GA for postfixation in 0.1 M PBS at pH 7.4. Prepare fresh
   from stock.

2.2 Tissue
Preparation and
Sectioning

1. 12% Gelatin: (Twee Torens). Prepare 12 w/v % gelatin as
   described in [4]. Briefly, resuspend 12 g gelatin in 100 mL
   0.1 M phosphate buffer. Stir for 5 min at RT (~21C). Incu-
   bate for 6 h at 60C and stir every 30 min. After the gelatin is
   dissolved, lower the temperature to 37C and add 100μLof
   20 w/v % sodium azide. Filter the solution in 5 mL vials. Place
   in the refrigerator until use (seeNote 3).


2. 2.3 M sucrose (D(+)-saccharose).

Fig. 1ISH-IEM of localized mRNAs in Drosophila oocytes. (a) High magnification visualization of endogenous
gurkenmRNA in stage 9 Drosophila oocyte by ISH-IEM on ultrathin frozen sections of a 4% paraformaldehyde
(PFA)-fixed stage 9 wild-type oocyte hybridized with a biotin-labeledgurkenRNA probe followed by a rabbit
anti-biotin antibody and protein A gold (PAG 15 nm) (arrows) and Me31B (PAG 10 nm) marking the P-bodies.
Note thatgurkenmRNA is at the edge of the P-bodies as described in [7]. (b) High magnification visualization
of endogenousbicoidmRNA in similar section but detected by a DIG labeledbicoidprobe followed by sheep
anti-DIG antibody, a rabbit anti-sheep antibody and PAG 5 nm. Note thatbicoidmRNA is preferentially found in
the interior of the P-bodies [7]. Scale bars: 500 nm

mRNA Detection by EM 179