- Rabbit anti-biotin (Rockland) to detect the biotinylated probe,
diluted 1:10,000 in 1% BSA in PBS. - To detect the DIG-labeled probe: Sheep anti-DIG Fab frag-
ments coupled to alkaline phosphatase (DIG-AP, 1:500,
Roche), or sheep anti-DIG Fab fragments coupled to HRP
(DIG-HRP, 1:1000, Roche), diluted in 1% BSA in PBS. - Rabbit anti-sheep IgG (1:750, Nordic) in 1% BSA in PBS.
- Protein-A Gold (PAG) conjugates diluted in 1% BSA in PBS
(see[4]). PAG can be purchased at the Department of Cell
Biology, UMC-Utrecht by subscription. For contact and infor-
mation: [email protected].
2.6 Contrasting 1. 4 w/v % uranyl acetate (UA) (SPI-CHEM) pH 4.0 prepared
and stored as described in [4].
- 2% UA-oxalate, pH 7.0. Mix 4% UA and 0.3 M oxalic acid in
1:1 ratio. Adjust pH to 7.0 with 25% NH 4 OH. - Uranyl acetate–methyl cellulose (UA-MC) pH 4.0. Mix 4% UA
with 2% MC in 1:9 ratio by stirring gently. The mixture can be
stored in the dark at 4C for up to 3 months. This reagent is
required for embedding and contrasting. - Wire loops with plastic handle. Loop diameter should slightly
exceed the diameter of the EM grids.
2.7 Viewing Transmission electron microscope (e.g., Jeol) to view sections.
Note that any electron microscope with an 80 kV power is
adequate.
3 Methods
3.1 Tissue Fixation From anaesthetize fattened 2-day-oldDrosophilafemale flies under
CO 2 , excise the whole ovaries in Ringer buffer using two pairs of
forceps and transfer them immediately to one of the two fixatives
(above) for 3–4 h at room temperature. When using 4% PFA,
continue with an overnight incubation at 4C. After fixation,
replace the fixative by 1% PFA in 0.1 M phosphate buffer at 4C
for storage (seeNote 2)[5, 8].
3.2 Embedding
and Ultrathin
Cryosectioning
- Dissect the ovaries into ovarioles and staged egg chambers in
gelatin and make blocks (as described in [5, 8]). - Incubate blocks in 2.3 M sucrose overnight at 4C in a rotator.
- Freeze blocks on roughened aluminum rivets with flat head in
liquid nitrogen.
mRNA Detection by EM 181