14 In Situ Hybridization for RNA: Nonradioactive Probe: RNA Probe
201
2. Wash sections in 2x SSC, 2x.
3. Treat the sections in RNase A (20/lg/ml) in 2x SSC for 30
mins at 37°e.
4. Wash sections in 2x SSC/50%FA for I hr at 50°e.
5. Wash sections in Ix SSC/50%FA for Ihr at 50°e.
1. Wash sections in PBS, 3x.
2. Immerse the sections in the blocking solution to block the
non-specific binding of antibody to the sections.
3. Apply anti-digoxigenin antibody conjugated with alkaline
phosphatase (1:500, Ihr-overnight) on the sections and incubate them for Ihr to overnight.
Note: Horseradish peroxidase can also be used.
4. Wash the sections with Tris HCI (10mM, pH 7.6)-buffered
saline (or PBS) containing 0.3% Tween 20, 3x.
5. Wash the sections with 100mM Tris HCI buffer (pH 9.5)/
100mM NaCl/50mM MgCl2 (phosphate should be removed).
6. Apply coloring solution (NBT/BCIP) on the sections. Put the
sections in a moist chamber in the dark. The reaction products appear in 30 mins to overnight.
7. Wash the sections in running water.
8. Fix the sections in 10% formalin for 10 mins.
9. Wash the sections in PBS for 30 mins to overnight. This may
be effective to avoid the precipitation of crystals on the reaction products.
10. Counterstain the sections with 2% methyl green for 10 mins.
11. Wash the sections with running water.
12. Air-dry the sections.
13. Drop xylene, and mount the sections with an embedding
substance.
Note: The original mounting method is aqueous mounting.
However, permanent specimens can be made by this method.
Immunohistochemical
detection of
signals
201
2. Wash sections in 2x SSC, 2x.
3. Treat the sections in RNase A (20/lg/ml) in 2x SSC for 30
mins at 37°e.
4. Wash sections in 2x SSC/50%FA for I hr at 50°e.
5. Wash sections in Ix SSC/50%FA for Ihr at 50°e.
1. Wash sections in PBS, 3x.
2. Immerse the sections in the blocking solution to block the
non-specific binding of antibody to the sections.
3. Apply anti-digoxigenin antibody conjugated with alkaline
phosphatase (1:500, Ihr-overnight) on the sections and incubate them for Ihr to overnight.
Note: Horseradish peroxidase can also be used.
4. Wash the sections with Tris HCI (10mM, pH 7.6)-buffered
saline (or PBS) containing 0.3% Tween 20, 3x.
5. Wash the sections with 100mM Tris HCI buffer (pH 9.5)/
100mM NaCl/50mM MgCl2 (phosphate should be removed).
6. Apply coloring solution (NBT/BCIP) on the sections. Put the
sections in a moist chamber in the dark. The reaction products appear in 30 mins to overnight.
7. Wash the sections in running water.
8. Fix the sections in 10% formalin for 10 mins.
9. Wash the sections in PBS for 30 mins to overnight. This may
be effective to avoid the precipitation of crystals on the reaction products.
10. Counterstain the sections with 2% methyl green for 10 mins.
11. Wash the sections with running water.
12. Air-dry the sections.
13. Drop xylene, and mount the sections with an embedding
substance.
Note: The original mounting method is aqueous mounting.
However, permanent specimens can be made by this method.
Immunohistochemical
detection of
signals