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- W2950197510 abstract "Objective Immunohistochemistry (IHC) is a widely used diagnostic technique in the Oral and Maxillofacial Pathology. Various variables affect results of the IHC technique. Therefore, standardization and optimization of the IHC technique are essential in generating reliable and reproducible results. The aim of this study was to determine the optimal conditions for IHC staining of multiple antibodies with minimal background. Findings In our study, we noticed that 2% hydrogen peroxide (H2O2) is most effective to block endogenous peroxidase activity. Formalin-fixed, Paraffin-embedded tissues from mice and human biopsies were subjected to IHC with three different monoclonal chimeric antibodies. Various combinations of antibody concentrations and incubation time were investigated. Two secondary antibody kits namely Vectastain Universal ABC-AP KIT (PK-6200) and Alkaline Phosphatase Universal (AK-5200) as well as two chromogen systems namely ImmPACT DAB EqV (Chromogen and peroxide) and Alkaline Phosphatase substrates were used. The optimal concentration of individual antibodies varied greatly (From 1 to 20 µg/ml) based on their affinity to the primary antigen. While the manufacturer instructions recommend 30 minutes incubation for all primary antibodies, we observed overnight incubation at 4oC obtained best results. The optimal counterstain with peroxidase (brown) substrate was Hematoxylin and Alkaline Phosphatase (blue) substrate was Nuclear Fast Red. Digital imaging parameters such as white balance, exposure time and file format were optimized. Evaluation of the IHC results was performed using the light microscope and digital imaging. Conclusion Overall, our results confirm careful validation of individual IHC technique is critical in obtaining consistent and reproducible results. Factors such as endogenous peroxidase blocking, antibody concentration and incubation time, chromogen system and counterstains are important parameters that should be optimized for individual studies." @default.
- W2950197510 created "2019-06-27" @default.
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- W2950197510 date "2019-07-01" @default.
- W2950197510 modified "2023-09-24" @default.
- W2950197510 title "OPTIMIZATION OF DIAGNOSTIC IMMUNOHISTOCHEMISTRY OF FORMALIN-fiXED, PARAffiN-EMBEDDED TISSUES" @default.
- W2950197510 doi "https://doi.org/10.1016/j.oooo.2019.02.071" @default.
- W2950197510 hasPublicationYear "2019" @default.
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