RNA Detection

4. Oocyte cultivation medium: full S2 cell culture medium, 15%
   fetal bovine serum (FBS), 1% streptomycin–penicillin antibio-
   tics mixture. Store at 4C until required. Before use, freshly
   add 200μg/mL human insulin.


5. A 3–9 well dissection plate.


6. Dumont #4 and #5 forceps.


7. Two sharpened tungsten needles mounted in handles.


8. Voltalef 10S halocarbon oil.

2.6 Wash-Free
Fluorescent in Situ
Hybridization (FISH)

1. Fixative: Mix a vial (10 mL) of 16% electron-microscopy grade
   paraformaldehyde (PFA) with 30 mL sterilized phosphate buff-
   ered saline (PBS, pH 7.4). Filter through 0.22 micron size
   particle filter to remove aggregates and other impurities.
   Store at 4C until required.


2. Wash buffer (IBEX): 10 mM Tris–HCl, pH 7.5–7.7, 100 mM
   KCl, 1 mM EDTA, 0.3 v/v % Triton-X-100.


3. 20 mg/mL Proteinase K (optional).


4. Ethylene carbonate (EC, optional).


5. 1.5 mL Eppendorf tubes.


6. Tube stands.


7. Micropipettes.


8. Nutator.


9. 37C rocking heating block.


10. 22 mm22 mm coverslips, nontreated glass slides to mount
    the specimen.


11. Coverslip sealant, e.g., transparent, nonglittering nail polish.

2.7 Microinjection 1. Eppendorf Femtotips II microinjection capillaries or

2. Borosilicate capillaries (e.g., Sutter 100-50-10).


3. Micropipette puller with 33 mm box filament to make
   microinjection capillaries (e.g., Sutter P-97).


4. Injection buffer (IB): 10 mM Tris–HCl, pH 7.5–7.7, 100 mM
   KCl, 1 mM MgCl 2.


5. Table-top microcentrifuge.


6. Microinjector (e.g., Eppendorf Femtojet series).


7. Incised plastic slide (seeFig. 1b) and 2222 mm coverslips.


8. Double sided tape.


9. Thin plastic sheet, e.g., used X-ray films.


10. 2–3 mm wide strips of Whatman 3 mm filter paper.

276 Jasmine Chamiolo et al.