Slides of human prostate cancer tissues were obtained and stained under ethics agreement HREC2012.275 (Royal Melbourne Hospital, Melbourne, Australia). Sections were dewaxed, rehydrated and antigen retrieval was performed for 15 min in Citrate buffer (pH 6.10). Endogenous peroxidase activity was blocked using 3% H2O2 in MetOH for 20min at room temperature. Slides were incubated for 2h in blocking buffer alone, followed by an overnight incubation at 4C in blocking buffer containing anti-TROP2 or isotype antibodies (BD Bioscience 551317, 1/500). Biotinylated anti-mouse secondary antibody (1/500, AbCAM) was incubated for 30 min at room temperature, followed by Tyramide Signal Amplification (TSA)(Perkin Elmer, Melbourne, Australia), revelation with diaminobenzidine (DAB) detection kit (AbCAM, Melbourne, Australia), Ethanol/Xylene dehydration and mounting. The percentage of cells considered as positive for TROP2 staining (cf example in Figure 1A ) was calculated after counting positive and total cell numbers in 5 randomly chosen fields of approximately 100 tumor cells.
Diaminobenzidine dab detection kit
Manufactured by Abcam
Sourced in Australia
The Diaminobenzidine (DAB) detection kit is a laboratory reagent used for the visualization of targeted proteins in various applications, such as immunohistochemistry, immunocytochemistry, and Western blotting. The kit contains a chromogenic substrate that, when oxidized by the enzyme horseradish peroxidase (HRP), produces a brown precipitate at the site of the target protein, allowing for its detection and localization.
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Lab products found in correlation
2 protocols using diaminobenzidine dab detection kit
1
Quantification of TROP2 Expression in Prostate Cancer
2
Immunohistochemistry of Paraffin-Embedded Tumors
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Freshly isolated tumors were fixed in 10% formalin and embedded in paraffin. For immunohistochemistry, tumor sections were deparaffinized, rehydrated, microwaved in 10 mmol/L citrate buffer for 30 min, and incubated in 0.3% Triton X-100 in PBS for 30 min. Sections were blocked using an Avidin-biotin blocking Kit (Abcam), and subsequently incubated with primary antibodies at the dilutions suggested by the manufacturer for 1 h, followed by secondary antibody for 30 min at room temperature. The diaminobenzidine (DAB) detection Kit (Abcam) was used to detect signals. Images were captured by light microscope (Leica DM4B).
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