Archived normal and tumour tissue biopsy slides were obtained from a subset of study participants (n=30) following approval by the Tissue and Archive Committee (Department of Pathology, London Health Sciences Centre). Antigen retrieval was performed with citrate buffer and slides were subsequently incubated with pre-immune serum or anti-OATP1B3 polyclonal antibody (1 : 200) followed by an avidin-biotin immunoperoxidase assay and developed using an AEC (3-Amino-9-ethylcarbazole) staining kit (Sigma-Aldrich) using a modified protocol (Lee et al, 2008 (link)). Nuclei were counterstained using Mayer's haematoxylin (Sigma-Aldrich). Scoring for OATP1B3 expression in normal, normal adjacent, and tumour tissue was performed independently by one pathologist (JP). Staining intensity for OATP1B3 was defined and evaluated using the following semi-quantitative previously published method: (0) no staining, (1) weakly positive, (2) moderately positive, and (3) strongly positive (Lee et al, 2008 (link)).
Aec 3 amino 9 ethylcarbazole staining kit
Manufactured by Merck Group
Sourced in Germany
The AEC (3-Amino-9-ethylcarbazole) staining kit is a laboratory product used for histochemical staining procedures. The kit provides the necessary reagents to perform a colorimetric staining reaction that can be used to visualize specific target molecules or cellular components in tissue samples.
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2 protocols using aec 3 amino 9 ethylcarbazole staining kit
1
Quantifying OATP1B3 Expression in Tissues
2
Quantifying HCMV Plaque Formation Under Bortezomib
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Cells (6.5 × 104) were seeded in 24-well plates and infected with HCMV isolate. After 1.5 h p.i. the inoculum was discarded, the cells were washed for three times with PBS and overlaid with 2 mL methylcellulose (Methocel MC, Fluka Analytical, München, Germany) containing DMEM with 10% FBS and 0.1–12.0 nM Bortezomib. After incubation for seven days at 37 °C, the cells were fixed using an -ethanol-acetone composition (95:5) and stained using an IE1/2 antibody (CCH2, DDG9 Dako) followed by treatment with the AEC (3-Amino-9-ethylcarbazole) staining kit (Sigma-Aldrich, Deisenhofen, Germany) as recommended by the manufacturer. Plaques were counted by the use of a light microscope (Axiovert 10, Carl Zeiss Microscopy GmbH, Jena, Germany). Compound effects were calculated by comparing compound-treated cells versus untreated cells.
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