Epitope retrieval and immunohistochemical (IHC) staining were performed as previously described.26 (link) A polyclonal rabbit antibody against full-length human GSTA4 with cross-reactivity to murine Gsta4 (Abnova, Walnut, CA, USA; H00002941-D01P, 1:100) and anti-4-HNE serum (Alpha Diagnostic International Inc, San Antonio, TX, USA; HNE11-S, 1:1,000) were used as primary antibodies. Goat anti-rabbit IgG-HRP conjugate (Life Technologies, Grand Island, NY, USA) was used as secondary antibody. Chromogenic color development was performed using 3,3′-diaminobenzidine enhanced liquid substrate (Sigma) and nuclei counterstained by Mayer’s hematoxylin (Sigma). IHC staining for GSTA4 in human tissues was scored using a combination of stain intensity (0, negative; 1, weak; 2, moderate; 3, strong) and overall number of positive cells per 20X field (0, none; 1, < 10%; 2, 10 – 50%; 3, >50% positive cells).
Protocol detail
Immunohistochemical Analysis of GSTA4
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anti-4-HNE serum
Goat anti-rabbit IgG-HRP conjugate
3,3′-diaminobenzidine enhanced liquid substrate
Mayer’s hematoxylin
Anti igg
Antibodies
Antibody
Cells
Chromogenic
Cross reactivity
Diagnostic
Epitope
Goat
Hematoxylin
Human
Murine
Nuclei
Rabbit
Serum
Stain
Tissues
Walnut
Weak
Corresponding Organization :
Other organizations : University of Oklahoma Health Sciences Center, Oklahoma City VA Health Care System
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Variable analysis
independent variables
- Epitope retrieval and immunohistochemical (IHC) staining methodology
- Primary antibodies: polyclonal rabbit antibody against full-length human GSTA4 with cross-reactivity to murine Gsta4, and anti-4-HNE serum
- Secondary antibody: Goat anti-rabbit IgG-HRP conjugate
- GSTA4 IHC staining intensity (0, negative; 1, weak; 2, moderate; 3, strong)
- Percentage of GSTA4 positive cells per 20X field (0, none; 1, < 10%; 2, 10 – 50%; 3, >50%)
- Chromogenic color development using 3,3′-diaminobenzidine enhanced liquid substrate
- Nuclei counterstaining with Mayer's hematoxylin
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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