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KO KOMUTA, TAKASHl KANEMATSU AND TAKEHlKO KOTl
2. Dry up for fresh frozen section: (S-6Mm) fresh frozen sections were dried up on gelatin coated slide glass.
3. Fixation: 4% PFA (10 mins 4°C)
4. PBS (RT, S mins x3)
5. Incubation: SO mM Tris/HCl(pH7.4)/S% non-fatdrymilk/SO
mM NaC1!( mM EDTA (RT, 1 hr)
6. Incubation: Dig-ds oligo-DNA (0.2S-2.0Mg/ml)/SO mM Trisl
HCl (pH7.4)/S% non-fat drymilk/SO mM NaC1!1 mM EDTA
(RT, overnight)
7. Washing: so mM Tris/HCl (pH7.4)/S% non-fat dry milk/SO
mMNaC1!1 mMEDTA (RT, ISminsx2) PBS (RT, ISminsx3)
8. Blocking: SOO Mg/ml sheep IgG/S% BSA/I00 Mg/ml salmon
testis DNA/100 Mg/ml yeast tRNA/NaN3/PBS (RT, 1 hr)
9. First antibody: HRP-sheep anti-Dig IgG(1: 100 )/S% BSA//1 00
Mg/ml salmon testis DNA/100 Mg/ml yeast tRNA/NaN3/PBS
(RT, overnight)
10. PBS (RT, IS mins x4)
11. Visualization: O.S mg/ml DAB/O.02S% CoClzI0.02% NiS04(NH4hS04 10.01 % H20 2 10.IM sodium phosphate buffer
(pH7.2) (RT, S mins)
The important points are explained as follows:
(a) Fixation
At the beginning of the experiments, the effects of fixation on the
reactivity in tissue sections were examined. When the sections
were fixed in 4% PFA for 10 mins at 4°C, the stainings were
strong. On the other hand, when the sections were fixed in acetone for 10 mins at 4°C, the stainings were very weak, and endogeneous peroxidase staining was remarkable. Consequently,
all subsequent studies were carried out using 4% PF A fixation
[ 10].
(b) Probe concentration
When the sections were reacted with the probe at various concentrations (0.5, 1.0, 2.S, or S Mg/ml), the staining intensity was
increased by increasing the probe concentration, reaching a plateu level at 1.0-2.S Mg/ml [10]. It is necessary to decide on the best
probe concentration for each individual probe.
KO KOMUTA, TAKASHl KANEMATSU AND TAKEHlKO KOTl
2. Dry up for fresh frozen section: (S-6Mm) fresh frozen sections were dried up on gelatin coated slide glass.
3. Fixation: 4% PFA (10 mins 4°C)
4. PBS (RT, S mins x3)
5. Incubation: SO mM Tris/HCl(pH7.4)/S% non-fatdrymilk/SO
mM NaC1!( mM EDTA (RT, 1 hr)
6. Incubation: Dig-ds oligo-DNA (0.2S-2.0Mg/ml)/SO mM Trisl
HCl (pH7.4)/S% non-fat drymilk/SO mM NaC1!1 mM EDTA
(RT, overnight)
7. Washing: so mM Tris/HCl (pH7.4)/S% non-fat dry milk/SO
mMNaC1!1 mMEDTA (RT, ISminsx2) PBS (RT, ISminsx3)
8. Blocking: SOO Mg/ml sheep IgG/S% BSA/I00 Mg/ml salmon
testis DNA/100 Mg/ml yeast tRNA/NaN3/PBS (RT, 1 hr)
9. First antibody: HRP-sheep anti-Dig IgG(1: 100 )/S% BSA//1 00
Mg/ml salmon testis DNA/100 Mg/ml yeast tRNA/NaN3/PBS
(RT, overnight)
10. PBS (RT, IS mins x4)
11. Visualization: O.S mg/ml DAB/O.02S% CoClzI0.02% NiS04(NH4hS04 10.01 % H20 2 10.IM sodium phosphate buffer
(pH7.2) (RT, S mins)
The important points are explained as follows:
(a) Fixation
At the beginning of the experiments, the effects of fixation on the
reactivity in tissue sections were examined. When the sections
were fixed in 4% PFA for 10 mins at 4°C, the stainings were
strong. On the other hand, when the sections were fixed in acetone for 10 mins at 4°C, the stainings were very weak, and endogeneous peroxidase staining was remarkable. Consequently,
all subsequent studies were carried out using 4% PF A fixation
[ 10].
(b) Probe concentration
When the sections were reacted with the probe at various concentrations (0.5, 1.0, 2.S, or S Mg/ml), the staining intensity was
increased by increasing the probe concentration, reaching a plateu level at 1.0-2.S Mg/ml [10]. It is necessary to decide on the best
probe concentration for each individual probe.