Formalin-fixed paraffin-embedded tissue sections were deparaffinized inxylene on microscopic slides. Antigen retrieval was performed by microwaving the sections in 10 mM citric acid buffer (pH 7.2). The primary antibodies used in this study were: anti-human IDH1-R132H monoclonal antibody (1:100, IBL Co., Ltd, Gumma, Japan) and anti-MGMT monoclonal antibody MT3.1 (1:200, Chemicon, Inc., Temecula, CA). The samples were incubated with the primary antibody overnight, followed by incubation with a biotinylated secondary antibody (1:500, Dako, Tokyo, Japan). The bound antibodies were visualized using the avidin biotin peroxidase complex method and diaminobenzidine tetrachloride (Santa Cruz Biotechnology, Inc.). To evaluate IDH1 staining, strong cytoplasmic staining in any number of cells was scored as positive. For MGMT scoring, the positive cells in a 200 × field (minimum of 1,000 nuclei) were counted, and the labeling index was expressed as a percentage of the labeled tumor cells. MGMT protein expression ≥10% was considered positive.
Avidin biotin peroxidase complex method
Manufactured by Santa Cruz Biotechnology
The Avidin biotin peroxidase complex method is a technique used in various biochemical and immunohistochemical applications. It utilizes the high-affinity binding interaction between avidin and biotin to amplify the signal in detection systems. This method involves the use of a biotinylated secondary antibody, which then binds to an avidin-biotin-peroxidase complex. The resulting complex can be detected using a chromogenic substrate, enabling the visualization and localization of the target molecule within a sample.
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2 protocols using avidin biotin peroxidase complex method
1
Immunohistochemical Analysis of IDH1 and MGMT in Tissue Sections
2
Quantitative Renal Morphology Analysis
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Articles displayed in microscopic fields (N = 10 fields per animal) were quantitated in a randomized and blinded fashion. Renal morphology was assessed with periodic acid-Schiff and Masson's trichrome staining (9, 10) . IHC was performed by the standard avidin-biotin-peroxidase complex method (Santa Cruz Biotechnologies, Santa Cruz, CA), as described elsewhere (9, 10) . The antibodies used included polyclonal p53 and aquaporin 1 (Santa Cruz Biotechnologies); anti-TGFβ1 antibody (R&D Systems, Burlington, Canada); monoclonal anticollagen type IV antibody (Chemicon International, Temecula, CA); polyclonal CD36 antibody (Abcam, Cambridge, MA); Fabp4 antibody (R&D Systems). Oxidative stress in vivo was assessed by dihydroethidium (Sigma-Aldrich, Oakville, Canada) staining in frozen kidney sections as reported previously (13) . The classic scoring of glomerulosclerosis (scale from 0 to 4) (39) and tubulo-interstitial injury (scale from 0 to 3) (40) was based on periodic acid-Schiff images.
The semi-quantitation of the relative staining values was performed by NIH Image J software (Bethesda, MD) (9, 10) . The images (N = 6-8 per animal) were analyzed and quantitated in a randomized and blinded fashion.
The semi-quantitation of the relative staining values was performed by NIH Image J software (Bethesda, MD) (9, 10) . The images (N = 6-8 per animal) were analyzed and quantitated in a randomized and blinded fashion.
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