18 Electron Microscopic in Situ Hybridization and its Combination
249
(c) Concentration of NaCl in the reaction mixture
It is known that an appropriate concentration of salt is required
to keep the specific interaction between protein and DNA. Also,
DNA binding proteins including transcription factors such as
steroid hormone receptors and cyclic AMP responsive element
binding proteins are extracted with a high-salt concentration
buffer [11]. In some trials, the concentration of N aCl in the reaction mixture was varied ranging from 50 to 200 mM. At a low
concentration of NaCl , e.g. 50 mM, the stainings were completelycovered with choromogen deposits. At a NaCl concentration
of 150 mM, very clear localization of the signals was obtained.
However, the staining was suddenly lost at the higher concentration of NaCl [12]. It is necessary to decide on the best concentration of NaCl for each individual trial.
In order to verify the specificity of the signal, we conducted var- Control
ious control expriments. When the probe was omitted from the experiments
reaction mixture, no nuclear staining was observed. Also, when a
section was reacted with the ds oligo-DNA probe in the presence
of an excess amount (100-fold) of unlabeled ds oligo-DNA pro be,
the nuclear staining was markedly decreased. In addition, when
a section was digested with RNase before the reaction with the
labeled ds oligo-DN A probe, the nuclear staining was not altered,
indicating that the nuclear staining was not due to nuclear RNA.
Applications
Localization of CRE-binding proteins
We synthesized an oligo-DNA with a consensus base sequence of
CRE (TGACGTCA) with three TTA repeats at the 5', end. Since
the CRE sequence is palindromic, the oligo-DNA was allowed to
self-anneal and form ds DNA with three TTA repeats at both
ends. The CRE ds-oligo-DNA was irradiated with UV light to
form haptenic T -T dimers. When the haptenized CRE dsoligo-DNA reacted with frozen sections fixed with 4% PFA in
PBS followed by enzyme immunohistochemical localization of
the T -T dimers, nuclei of intestinal epithelial cells and brain cells
were heavily stained [10].
249
(c) Concentration of NaCl in the reaction mixture
It is known that an appropriate concentration of salt is required
to keep the specific interaction between protein and DNA. Also,
DNA binding proteins including transcription factors such as
steroid hormone receptors and cyclic AMP responsive element
binding proteins are extracted with a high-salt concentration
buffer [11]. In some trials, the concentration of N aCl in the reaction mixture was varied ranging from 50 to 200 mM. At a low
concentration of NaCl , e.g. 50 mM, the stainings were completelycovered with choromogen deposits. At a NaCl concentration
of 150 mM, very clear localization of the signals was obtained.
However, the staining was suddenly lost at the higher concentration of NaCl [12]. It is necessary to decide on the best concentration of NaCl for each individual trial.
In order to verify the specificity of the signal, we conducted var- Control
ious control expriments. When the probe was omitted from the experiments
reaction mixture, no nuclear staining was observed. Also, when a
section was reacted with the ds oligo-DNA probe in the presence
of an excess amount (100-fold) of unlabeled ds oligo-DNA pro be,
the nuclear staining was markedly decreased. In addition, when
a section was digested with RNase before the reaction with the
labeled ds oligo-DN A probe, the nuclear staining was not altered,
indicating that the nuclear staining was not due to nuclear RNA.
Applications
Localization of CRE-binding proteins
We synthesized an oligo-DNA with a consensus base sequence of
CRE (TGACGTCA) with three TTA repeats at the 5', end. Since
the CRE sequence is palindromic, the oligo-DNA was allowed to
self-anneal and form ds DNA with three TTA repeats at both
ends. The CRE ds-oligo-DNA was irradiated with UV light to
form haptenic T -T dimers. When the haptenized CRE dsoligo-DNA reacted with frozen sections fixed with 4% PFA in
PBS followed by enzyme immunohistochemical localization of
the T -T dimers, nuclei of intestinal epithelial cells and brain cells
were heavily stained [10].