store in the dark. The buffer should be discarded when it turns
yellow.
17. 2Â RNA-loading dye: 95% Formamide, 0.025% SDS, 0.025%
bromophenol blue, 0.025% xylene cyanol FF, 0.025% ethidium
bromide, 0.5 mM EDTA.
18. Agarose.
19. 37% Formaldehyde.
20. 10Â 3-(N-morpholino)propanesulfonic acid (MOPS): 0.4 M
MOPS (pH 7.0), 0.1 M sodium acetate, 0.01 M EDTA
(pH 8.0). All dilutions should be prepared with RNase-free
double-distilled water.
2.4 Whole-Mount In
Situ Hybridization
(WISH)
1. 3% (v/v) H 2 O 2 in double-distilled water.
2. 10% (w/v) Formaldehyde stock solution: Prepare a 10% (w/v)
solution of paraformaldehyde in RNase-free double-distilled
water. Heat solution (!60
C) carefully and add a few drops
of 1 N NaOH until the solution becomes clear (pH ~8); check
the pH using a pH paper indicator and store the solution in
single-use aliquots at À20
C. (Cautions: Vapors are toxic;
work under a fume hood).
3. 10Â Phosphate-buffered saline (PBS): 1.3 M NaCl, 70 mM
Na 2 HPO 4 , 30 mM NaH 2 PO 4 , pH 7.4, treated with DEPC.
Prepare all dilutions with RNase-free double-distilled water.
4. Fixative: 4% (w/v) Formaldehyde (from 10% stock solution),
15% (v/v) dimethyl sulfoxide (DMSO), 0.1% (v/v) Tween
® 20,
in 1Â PBS.
5. n-Heptan.
6. PBS-T: 1Â PBS, 0.1% (v/v) Tween
® 20.
7. HistoClear/ethanol (1/1).
8. Freshly prepared 3% (v/v) H 2 O 2 in ethanol.
9. 20Â Saline sodium citrate buffer (SSC): 3 M NaCl, 0.3 M
sodium citrate; adjust the pH to 7.0 with HCl and treat with
DEPC. All dilutions should be prepared with RNase-free double-distilled water.
10. 75% (v/v) Ethanol in RNase-free double-distilled water.
11. 80% (v/v) Ethanol in RNase-free double distilled water.
12. 50% (v/v) Ethanol in PBS.
13. 25% (v/v) Ethanol in PBS.
14. Proteinase K stock solution: 25 mg/mL Proteinase K in
RNase-free double-distilled water (store in single-use aliquots
at À20
C).
8
Thomas Dresselhaus and Andrea Bleckmann