184
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
Required
solutions
follows. First, contamination of RNase should be avoided at all
steps of the first day. Second, sections must not be dried at any
step once the procedure of in situ hybridization is started. If the
sections are dry, a non-specific signal is obtained. Finally, to the
beginner, be careful when handling a very small amount of the
reagent, especially on the first day.
Materials
- O.C.T compo.und (Miles Inc., Elkhardt, IN)
- Diethylpyrocarbonate (Sigma)
- 3-aminopropyltriethoxysilane (Sigma)
- Paraformaldehyde (Merk 104005)
- Digoxigenin oligonucleotide tailing kit (Boehringer Mannheim 1417231, Germany)
- Diethylpyrocarbonate-treated phosphate buffered saline
(PBS)
- Proteinase K (Sigma P-4914)
- Salmon testis DNA (Sigma)
- Bovine serum albumin (BSA, Sigma)
- Ficoll (Sigma)
- Polyvinylpyrrolidone (Sigma)
- AG 501-X8 Resin (BIO-RAD 142-6425)
- Incubation chamber
- BRIJ 35 (23 Lauryl ether, Sigma)
- Monoclonal anti-digoxigenin antibody (Boehringer Mannheim 1333 062)
- Normal swine serum (DAKO)
- Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse
antibody (Dako P260)
- HRP-conjugated swine anti-rabbit antibody (Dako Z113)
- Diethylpyrocarbonate (DEPC)-treated water:
Add 0.1 % DEPC to distilled water and stir it well for at least
one hour. Autoclave the solution prior to use.
Note: To obtain the signal, preservation of mRNA in sections is
essential. It is necessary to minimize the activity of RN ase in all
steps of in situ hybridization as well as Northern blotting or polymerase chain reaction. All solutions should be prepared using
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
Required
solutions
follows. First, contamination of RNase should be avoided at all
steps of the first day. Second, sections must not be dried at any
step once the procedure of in situ hybridization is started. If the
sections are dry, a non-specific signal is obtained. Finally, to the
beginner, be careful when handling a very small amount of the
reagent, especially on the first day.
Materials
- O.C.T compo.und (Miles Inc., Elkhardt, IN)
- Diethylpyrocarbonate (Sigma)
- 3-aminopropyltriethoxysilane (Sigma)
- Paraformaldehyde (Merk 104005)
- Digoxigenin oligonucleotide tailing kit (Boehringer Mannheim 1417231, Germany)
- Diethylpyrocarbonate-treated phosphate buffered saline
(PBS)
- Proteinase K (Sigma P-4914)
- Salmon testis DNA (Sigma)
- Bovine serum albumin (BSA, Sigma)
- Ficoll (Sigma)
- Polyvinylpyrrolidone (Sigma)
- AG 501-X8 Resin (BIO-RAD 142-6425)
- Incubation chamber
- BRIJ 35 (23 Lauryl ether, Sigma)
- Monoclonal anti-digoxigenin antibody (Boehringer Mannheim 1333 062)
- Normal swine serum (DAKO)
- Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse
antibody (Dako P260)
- HRP-conjugated swine anti-rabbit antibody (Dako Z113)
- Diethylpyrocarbonate (DEPC)-treated water:
Add 0.1 % DEPC to distilled water and stir it well for at least
one hour. Autoclave the solution prior to use.
Note: To obtain the signal, preservation of mRNA in sections is
essential. It is necessary to minimize the activity of RN ase in all
steps of in situ hybridization as well as Northern blotting or polymerase chain reaction. All solutions should be prepared using