Performance of immunohistochemistry was as set forth [26 (link)]. Dewaxing of paraffin sections was in xylene and rehydration was via an alcohol gradient. After antigenic repair, seal of all sections was in an avidin/biotin blocking buffer (Vector Laboratories) and then in 3% Bovine serum albumin. Incubation of the sections was with primary antibody Ki-67 (ab15580, Abcam), CD206 (18,704-1-AP, Protein-tech) and CD163 (ab182422, Abcam). Protein staining was via Diaminobenzidine substrate kit (Maixin Biotech, Kit-9710). Counterstain of the samples was with hematoxylin. Gain of immunohistochemical images was via a forward microscope (Olympus BX51). Brown staining clarified immunoreactivity of the sample.
Diaminobenzidine substrate kit
Manufactured by Maixin Group
The Diaminobenzidine substrate kit is a laboratory reagent used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) techniques. It provides a chromogenic substrate that reacts with horseradish peroxidase (HRP) to produce a brown colored product, enabling visualization and detection of target analytes.
Automatically generated - may contain errors
2 protocols using diaminobenzidine substrate kit
1
Immunohistochemistry Protocol for Protein Staining
2
Immunohistochemical Analysis of BAP18 in Prostate Tissue
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Sections of prostate tissue specimens were prepared from clinical prostatectomy specimens in the First Hospital of China Medical University. The procedure was performed as previously described (28 (link),31 (link)). Tissue sections of formalin-fixed, paraffin embedded tissue blocks were dewaxed in xylene, rehydrated in decreasing concentrations of ethanol, and blocked for endogenous peroxidase activity using 3% hydrogen peroxide. Antigen retrieval was carried out in citrate buffer (pH 6.0) at 130°C for 2 min. Then, non-specific antibody binding was blocked by incubating with goat serum for 15 min at room temperature. Slides were incubated with anti-BAP18 (1:100) overnight at 4°C. After washing, slides were incubated with biotinylated goat anti-rabbit/mouse immunoglobulin, followed by incubation with avidin DH-biotinylated horseradish peroxidase complex (UltraSensitiveTM SP(Mouse/Rabbit)IHC Kit, Maixin Bio). The sections were developed with the diaminobenzidine substrate kit (Maixin Bio) and counterstained with hematoxylin. Images were taken with an Olympus microscope. This study was approved by the Ethics Committee of the Medical Faculty of the China Medical University.
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