Immunohistochemical techniques were utilized for assessment of the presence of Bcl-2, Bax, and cleaved caspase-3 [18 ,19 (link)]. Briefly, endogenous peroxidases in the sections were blocked with H2O2, and the sections were then blocked with bovine serum albumin and normal goat serum. Rabbit polyclonal anti-Bcl-2, anti-Bax (1:50 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA), or anti-cleaved caspase-3 primary antibody (1:50 dilution, Cell Signaling, Beverly, MA) was added, and the sections were stained using a Vectastain Elite ABC kit (Vector Laboratories, Burlington, ON), in which color was developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB; Roche, Mannheim, Germany). The sections were evaluated at a 200× magnification. The intensity of DAB staining was measured using Image J software. Relative values for the intensity were used by setting the value for the sham group at 1.
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Immunohistochemical Analysis of Apoptosis Markers
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anti-Bax
anti-cleaved caspase-3 primary antibody
Vectastain Elite ABC kit
3,3'-diaminobenzidine tetrahydrochloride (DAB;
Anti antibody
Bcl 2
Bovine serum albumin
Caspase
Caspase 3
Dilution
Goat
H2o2
Peroxidases
Rabbit
Serum
Vector
Corresponding Organization :
Other organizations : Catholic University of Daegu
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Variable analysis
independent variables
- Bcl-2
- Cleaved caspase-3
- Intensity of DAB staining
- Endogenous peroxidases
- Bovine serum albumin
- Normal goat serum
- Magnification (200x)
- Sham group (used as a reference for relative values of DAB staining intensity)
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