246
KO KOMUTA, TAKASHl KANEMATSU AND TAKEHlKO KOrl
blue were subjected to SDS-PAGE with a 4-20 % gradient gel in
duplicate. One slab of the separated proteins was electrophoretically transferred onto nylon membranes according to the user
manual of immunoblotting system (Daiichi Pure Chern. Co., Japan), and one of them was stained with the silver staining for
visualization of protein bands. The molecular weight markers
were as follows; bovine skeletal muscle myoglobin (17.2 kD), bovine erythrocyte carbonic anhydrase (30 kD), rabbit skeletal
muscle aldolase (42.4 kD), bovine serum albumin (66.3 kD),
E. coli -galactosidase (116.2 kD) and rabbit skeletal muscle myosin myosin (200 kD).
The blotted nylon membrane was processed according to
Silva et al. [8] with a slight modification. Briefly, the blot was
incubated with a preincubation solution containing 5 % nonfat dry milk dissolved in 10 mM Tris/HCI (pH 7.4) buffer containing 50 mM NaCI and 1 mM EDTA for overnight at 4°C. Then the
blot was reacted with 1 /lg/ml Dig-labeled or T -T dimerized ds
oligo-DNA dissolved in the preincubation solution for 3 hrs at
. RT. After successive washings with the preincubation solution
(twice, 1 hr each) and PBS (3 times, 1 hr each), the blot was incubated for 1 hr in a blocking solution that contained 5 % BSA
and 500 /lg/ml normal sheep or goat IgG in PBS. The reaction
with HRP-linked sheep anti-Dig IgG (Fab), which was diluted
at 1:800 with the blocking solution, was performed for 1 hr.
The T -T dimers were detected immunohistochemically using
rabbit anti-(T-T dimer) IgG and HRP-Fab (anti-R IgG) in succession. As a chromogen solution of HRP, a mixture of DAB,
H20 2, cobalt chloride and nickel ammonium sulfate was used
[9] .
To confirm the sequence specificity in the reaction oflabeled
ds oligo-DNA probes with the blotted proteins, the blot was reacted with 1 /lg/ml probe in the presence of an excess amount
(100 - 450-fold) of the unlabeled ds oligo-DNA.
Tissue In our past system, we used the fresh frozen sections alone. Impreparation mediately after resection, specimens (size;5-10 mm) were embedded in OCT compound and frozen under dry ice added ethanol.The frozen sections were stored under -80 a C.
Southwestern The procedure of Southwestern histochemistry using Thyminehistochemistry thymine (T -T) dimers is as follows:
KO KOMUTA, TAKASHl KANEMATSU AND TAKEHlKO KOrl
blue were subjected to SDS-PAGE with a 4-20 % gradient gel in
duplicate. One slab of the separated proteins was electrophoretically transferred onto nylon membranes according to the user
manual of immunoblotting system (Daiichi Pure Chern. Co., Japan), and one of them was stained with the silver staining for
visualization of protein bands. The molecular weight markers
were as follows; bovine skeletal muscle myoglobin (17.2 kD), bovine erythrocyte carbonic anhydrase (30 kD), rabbit skeletal
muscle aldolase (42.4 kD), bovine serum albumin (66.3 kD),
E. coli -galactosidase (116.2 kD) and rabbit skeletal muscle myosin myosin (200 kD).
The blotted nylon membrane was processed according to
Silva et al. [8] with a slight modification. Briefly, the blot was
incubated with a preincubation solution containing 5 % nonfat dry milk dissolved in 10 mM Tris/HCI (pH 7.4) buffer containing 50 mM NaCI and 1 mM EDTA for overnight at 4°C. Then the
blot was reacted with 1 /lg/ml Dig-labeled or T -T dimerized ds
oligo-DNA dissolved in the preincubation solution for 3 hrs at
. RT. After successive washings with the preincubation solution
(twice, 1 hr each) and PBS (3 times, 1 hr each), the blot was incubated for 1 hr in a blocking solution that contained 5 % BSA
and 500 /lg/ml normal sheep or goat IgG in PBS. The reaction
with HRP-linked sheep anti-Dig IgG (Fab), which was diluted
at 1:800 with the blocking solution, was performed for 1 hr.
The T -T dimers were detected immunohistochemically using
rabbit anti-(T-T dimer) IgG and HRP-Fab (anti-R IgG) in succession. As a chromogen solution of HRP, a mixture of DAB,
H20 2, cobalt chloride and nickel ammonium sulfate was used
[9] .
To confirm the sequence specificity in the reaction oflabeled
ds oligo-DNA probes with the blotted proteins, the blot was reacted with 1 /lg/ml probe in the presence of an excess amount
(100 - 450-fold) of the unlabeled ds oligo-DNA.
Tissue In our past system, we used the fresh frozen sections alone. Impreparation mediately after resection, specimens (size;5-10 mm) were embedded in OCT compound and frozen under dry ice added ethanol.The frozen sections were stored under -80 a C.
Southwestern The procedure of Southwestern histochemistry using Thyminehistochemistry thymine (T -T) dimers is as follows: