Tissue sections were deparaffinized in xylene and microwaved in 10 mM sodium citrate buffer (pH 6.0) to unmask the epitopes. Endogenous peroxidase activity was blocked by incubating for 10 min with 3% hydrogen peroxide in methanol. Immunohistochemical staining for Aurora B (1:200), Ki-67 (1:200) or p-histone H3 Ser10 (1:200) was performed by using the indirect avidin biotin-enhanced horseradish peroxidase method according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). After developing with 3,3’-diaminobenzidine, all sections were counterstained with hematoxylin and observed by microscope (200× magnification). Quantitative analysis of immunohistochemical staining was performed using the Image-Pro Plus software (version 6.2) program (Media Cybernetics, Inc., Rockville, MD).
Indirect avidin biotin enhanced horseradish peroxidase method
Manufactured by Vector Laboratories
Sourced in United States
The Indirect avidin biotin-enhanced horseradish peroxidase method is a laboratory technique used to amplify signals in various immunoassays. It employs a multi-step detection system that utilizes the high affinity between avidin and biotin to enhance the signal generated by horseradish peroxidase, an enzyme commonly used as a reporter in these types of assays.
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4 protocols using indirect avidin biotin enhanced horseradish peroxidase method
1
Quantitative Immunohistochemical Analysis
2
Immunohistochemical Analysis of Tumor Tissues
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Animal tumor tissues were embedded in paraffin and subjected to immunohistochemistry staining. Tissues were deparaffinized and hydrated and then permeabilized in 0.5% Triton X-100/1 × PBS for 10 min. Immunohistochemical staining for Ki-67 (1:150), phosphorylated (p)-EGFR (1:400), p-ERK1/2 (1:400) or p-Akt (1:50) was performed using the indirect avidin biotin-enhanced horseradish peroxidase method according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA). After developing, all sections were observed by microscope (20x) and analyzed using the Image-Pro Premier software off line (v.9.0) program (Media Cybernetics).
3
Immunohistochemical Analysis of Esophageal Cancer
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Esophageal cancer tissues and animal tissues were embedded in paraffin and subjected to immunohistochemistry. Tissues were deparaffinized and hydrated and then permeabilized with 0.5% Triton X-100/1 × PBS for 10 min. Immunohistochemical staining for Ki-67 (1:100), phosphorylated (p)-histone H3 (1:100) or cleaved caspase 3 (1:200) was performed using the indirect avidin biotin-enhanced horseradish peroxidase method according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA). After developing, all sections were observed by microscope (100×) and analyzed using Image-Pro Plus software (v. 6.2) program (Media Cybernetics).
4
BRAF Immunohistochemical Staining Protocol
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Tumor tissues were embedded in paraffin and subjected to IHC staining. Tissues were deparaffinized and hydrated and then permeabilized in 0.5% Triton X-100 in PBS for 10 mins. IHC staining for BRAF-antibody (1:250) was performed using the indirect avidin biotin-enhanced horseradish peroxidase method according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA, USA). After developing, all sections were observed by microscope (20x) and analyzed using the Image-Pro Premier software offline (v.9.0) program (Media Cybernetics).
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