Immunohistochemical staining was performed as described previously [27 (link)]. The cross-sectional heart tissue fixed with 10% neutral buffered
formalin was embedded in paraffin, and thin sliced section (4 µm) of the
heart tissue was made. After the deparaffinization, the sections were heated with
microwave for activation of antigens in sodium citrate buffer (pH 6.0), and endogenous
peroxidase activity was blocked by treating with 3% H2O2 for 10 min.
Then, the sections were blocked with 5% normal goat serum and incubated with primary
antibody against arresten (1:200 dilution), canstatin (1:200 dilution) or cathepsin S
(1:50 dilution) at 4°C overnight. After washing, the sections were incubated in
biotinylated link (Dako, Glostrup, Denmark) for 10 min and treated with
streptavidin-horseradish peroxidase (Dako) for 10 min at room temperature. Then,
expression of canstatin and arresten was visualized by a liquid 3,3′-Diaminobenzidine
(DAB) + substrate chromogen system (Dako). The images were obtained using a light
microscope (BX-51, OLYMPUS) equipped with a microscope digital camera (DP74, OLYMPUS). The
positive area was quantified by using ImageJ software (National Institutes of Health,
Bethesda, MD, U.S.A.).
formalin was embedded in paraffin, and thin sliced section (4 µm) of the
heart tissue was made. After the deparaffinization, the sections were heated with
microwave for activation of antigens in sodium citrate buffer (pH 6.0), and endogenous
peroxidase activity was blocked by treating with 3% H2O2 for 10 min.
Then, the sections were blocked with 5% normal goat serum and incubated with primary
antibody against arresten (1:200 dilution), canstatin (1:200 dilution) or cathepsin S
(1:50 dilution) at 4°C overnight. After washing, the sections were incubated in
biotinylated link (Dako, Glostrup, Denmark) for 10 min and treated with
streptavidin-horseradish peroxidase (Dako) for 10 min at room temperature. Then,
expression of canstatin and arresten was visualized by a liquid 3,3′-Diaminobenzidine
(DAB) + substrate chromogen system (Dako). The images were obtained using a light
microscope (BX-51, OLYMPUS) equipped with a microscope digital camera (DP74, OLYMPUS). The
positive area was quantified by using ImageJ software (National Institutes of Health,
Bethesda, MD, U.S.A.).