Monoclonal antibodies (Abs) against human P-gp (1:200; clone F4; Sigma-Aldrich Co., LLC, St. Louis, MO, USA) and LRP (1:100; clone 1032; Beijing Golden Bridge Biotechnology Company Ltd., Beijing, China) were used as primary antibodies. Biotin-streptavidin-peroxidase staining with 3, 3'-diaminobenzidine-tetrahydrochloride detection was used. Tumor cells with staining in the cell membrane or cytoplasm were considered to be positive. Each slide was graded blindly according to the percentage of positive tumor cells. The immunoreactivity of P-gp and LRP was scored as negative (-) when the proportion of positive tumor cells was less than 10% or as positive (+) when the number of positive tumor cells was between 10% and 100%.
Clone f4
Manufactured by Merck Group
Sourced in United States
The Clone F4 is a laboratory equipment product designed for cell culture applications. It serves as a tool for the cloning and propagation of genetic material. The core function of the Clone F4 is to provide a controlled environment for the cultivation and replication of cells or organisms.
Automatically generated - may contain errors
2 protocols using clone f4
1
Immunohistochemical Analysis of P-gp and LRP
2
Quantification of P-glycoprotein Expression
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Western blot analysis of P-gp expression was performed as described previously (24) using monoclonal anti-P-gp (ABCB1) antibody produced in mouse, clone F4 (1 : 1000; Sigma-Aldrich) and polyclonal anti-a-tubulin (1 : 2000; Cell Signaling Technology, Danvers, MA, USA). The signal was detected using a horseradish peroxidase-conjugated secondary antibody (1 : 3000; Dako, Glostrup, Denmark). Products were visualised using an enhanced chemiluminescence (ECL; Amersham, Little Chalfont, UK).
Flow cytometric analysis of P-gp expression P-gp expression was measured using UIC2 (Pgp, Beckman Coulter, Miami, USA) monoclonal antibody conjugated with phycoerythrin (UIC2-PE) according to the manufacturer's instruction. The fluorescence of the cells was measured and analysed by flow cytometry. Protein expression was quantified as the median fluorescence intensity shift (MFI). Phycoerythrin conjugated isotype IgG2a was used as a control. The fluorescence of the cells was analysed by flow cytometry (Cytomics FC500, Beckman Coulter). ABCB1 expression was determined by the ratio of the mean fluorescence intensity (MFI) shift of UIC2-PE antibody to isotype control (UIC2-PE/IgG2a-PE). For each sample, 10 000 events were collected.
Flow cytometric analysis of P-gp expression P-gp expression was measured using UIC2 (Pgp, Beckman Coulter, Miami, USA) monoclonal antibody conjugated with phycoerythrin (UIC2-PE) according to the manufacturer's instruction. The fluorescence of the cells was measured and analysed by flow cytometry. Protein expression was quantified as the median fluorescence intensity shift (MFI). Phycoerythrin conjugated isotype IgG2a was used as a control. The fluorescence of the cells was analysed by flow cytometry (Cytomics FC500, Beckman Coulter). ABCB1 expression was determined by the ratio of the mean fluorescence intensity (MFI) shift of UIC2-PE antibody to isotype control (UIC2-PE/IgG2a-PE). For each sample, 10 000 events were collected.
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