15. 10Â Glycine solution: 200 mg/mL Glycine in RNase-free 10x
PBS. Store aliquots at À20
C. Dilutions should be prepared
with RNase-free double-distilled water.
16. 50 mg/mL Heparin stock solution (store at À20
C).
17. RNase-free fragmentized sperm DNA: Purify a fragmentized
DNA (~500 bp) solution by phenol-chloroform-isoamyl alcohol extraction followed by a chloroform extraction and an
ethanol precipitation. Resuspend in RNase-free double-distilled water. Adjust DNA concentration to 10–12 mg/mL
using RNase-free double-distilled water.
18. Pre-hybridization solution: 50% (v/v) Formamide, 5Â SSC,
0.1% (v/v) Tween
® 20, and 0.1 mg/mL heparin in RNase-free
double-distilled water.
19. Washing solution 1: 50% (v/v) formamide, 2Â SSC, 0.1%
(v/v) Tween
®
20 in RNase-free double-distilled water.
20. Washing solution 2: 2Â SSC, 0.1% (v/v) Tween
® 20 in RNasefree double-distilled water.
21. Washing solution 3: 0.2Â SSC, 0.1% (v/v) Tween
® 20 in
RNase-free double-distilled water.
22. Blocking solution: 3% (w/v) Bovine serum albumin (BSA) in
PBS-T. Prepare freshly before use.
23. Anti-digoxigenin-AP-conjugated antibody (e.g., 1:2000 of the
antibody 11093274910 from Roche in blocking solution).
24. Anti-digoxigenin-HRP-conjugated antibody (e.g., 1:1000 of
the antibody NEF832001EA from Perkin Elmer in blocking
solution).
25. Anti-fluorescein-HRP-conjugated antibody (e.g., 1:1000 of
the antibody NEF710001EA from Perkin Elmer in blocking
solution).
26. 1 M MgCl 2 (DEPC treated; see Subheading 3.1, step 4).
27. 2 M Levamisole in RNase-free double-distilled water (store
aliquots at À20
C).
28. Alkaline phosphatase (AP) buffer: 0.1 M Tris–HCl (pH 9.5),
0.1 M NaCl, 50 mM MgCl 2 (freshly added), 0.1% (v/v)
Tween
®
20.
29. 50 mg/mL Nitroblue tetrazolium chloride (NBT) stock solution (store at À20
C).
30. 50 mg/mL 5-Bromo-4-chloro-3-indoxyl phosphate (BCIP)
stock solution (store at À20
C).
31. AP staining solution: AP buffer containing 2 mM levamisole,
220 μg/mL NBT, and 180 μg/mL BCIP.
32. TNT buffer: 0.1 M Tris–HCl (pH 7.5), 0.15 M NaCl, 0.05%
(v/v) Tween
®
20.
RNA-FISH
9
PBS. Store aliquots at À20
C. Dilutions should be prepared
with RNase-free double-distilled water.
16. 50 mg/mL Heparin stock solution (store at À20
C).
17. RNase-free fragmentized sperm DNA: Purify a fragmentized
DNA (~500 bp) solution by phenol-chloroform-isoamyl alcohol extraction followed by a chloroform extraction and an
ethanol precipitation. Resuspend in RNase-free double-distilled water. Adjust DNA concentration to 10–12 mg/mL
using RNase-free double-distilled water.
18. Pre-hybridization solution: 50% (v/v) Formamide, 5Â SSC,
0.1% (v/v) Tween
® 20, and 0.1 mg/mL heparin in RNase-free
double-distilled water.
19. Washing solution 1: 50% (v/v) formamide, 2Â SSC, 0.1%
(v/v) Tween
®
20 in RNase-free double-distilled water.
20. Washing solution 2: 2Â SSC, 0.1% (v/v) Tween
® 20 in RNasefree double-distilled water.
21. Washing solution 3: 0.2Â SSC, 0.1% (v/v) Tween
® 20 in
RNase-free double-distilled water.
22. Blocking solution: 3% (w/v) Bovine serum albumin (BSA) in
PBS-T. Prepare freshly before use.
23. Anti-digoxigenin-AP-conjugated antibody (e.g., 1:2000 of the
antibody 11093274910 from Roche in blocking solution).
24. Anti-digoxigenin-HRP-conjugated antibody (e.g., 1:1000 of
the antibody NEF832001EA from Perkin Elmer in blocking
solution).
25. Anti-fluorescein-HRP-conjugated antibody (e.g., 1:1000 of
the antibody NEF710001EA from Perkin Elmer in blocking
solution).
26. 1 M MgCl 2 (DEPC treated; see Subheading 3.1, step 4).
27. 2 M Levamisole in RNase-free double-distilled water (store
aliquots at À20
C).
28. Alkaline phosphatase (AP) buffer: 0.1 M Tris–HCl (pH 9.5),
0.1 M NaCl, 50 mM MgCl 2 (freshly added), 0.1% (v/v)
Tween
®
20.
29. 50 mg/mL Nitroblue tetrazolium chloride (NBT) stock solution (store at À20
C).
30. 50 mg/mL 5-Bromo-4-chloro-3-indoxyl phosphate (BCIP)
stock solution (store at À20
C).
31. AP staining solution: AP buffer containing 2 mM levamisole,
220 μg/mL NBT, and 180 μg/mL BCIP.
32. TNT buffer: 0.1 M Tris–HCl (pH 7.5), 0.15 M NaCl, 0.05%
(v/v) Tween
®
20.
RNA-FISH
9