Following exposure to 1 µmol L−1 BK, NT or SP for 60 minutes, the cells were quickly washed with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Fisher Scientific, Waltham, MA, USA) for 5 minutes and blocked for 30 minutes (10% goat serum and 0.2% Triton-X100 in PBS). Then, the BMEC monolayers were incubated with mouse-anti human claudin-5 (dilution 1:100, ThermoFisher; RRID: AB_2533200) or mouse-anti human occludin (dilution 1:100, ThermoFisher; AB_2533101) antibodies overnight at 4°C. Primary antibodies were detected using a goat-anti mouse IgG Alexa Fluor® 555-conjugated secondary anti-body (ThermoFisher). In experiments involving F-actin, freshly fixed BMEC monolayers were stained with 0.1 mg mL−1 fluorescein isothiocyanate-conjugated phalloidin (Sigma-Aldrich) for 30 minutes. All images were acquired using a DMi8 inverted epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). Images were analysed using IMAGEJ (NIH, Bethesda, MD, USA).
Ab 2533200
Manufactured by Thermo Fisher Scientific
The AB_2533200 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed for general laboratory use, but a detailed product description is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab 2533101, by Thermo Fisher Scientific (2 mentions)
Fluorescein isothiocyanate conjugated phalloidin, by Merck Group (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dmi 8 inverted epi uorescence microscope, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
2 protocols using ab 2533200
1
Immunofluorescent Localization of Tight Junction Proteins
2
Immunofluorescence Staining of Glucose Transporters
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Cells were quickly washed with ice-cold PBS and xed with 100% cold methanol or 4% paraformaldehyde, followed by blocking in PBSG (PBS with 10% normal goat serum (ThermoFisher)) with 0.2% Triton-X100 (Sigma-Aldrich) for 1h at room temperature. Cells were incubated in presence of primary antibodies targeting GLUT-1 (SPM498 clone, RRID:AB_10979643, 1:100; ThermoFisher), GLUT-3 (RRID:AB_10694437, 1:100;; ThermoFisher), GLUT4 (RRID:AB_11153908, 1:100; ThermoFisher), occludin (RRID: AB_2533101, 1:100, ThermoFisher), and claudin-5 (AB_2533200, 1:100, ThermoFisher) overnight at 4°C, followed by detection using Alexa-Fluor-conjugated secondary antibodies (ThermoFisher) for 1h at room temperature. Cells were observed using Leica DMI8 inverted epi uorescence microscope (Leica Microsystems, Buffalo Grove, IL, USA). Micrograph images were processed and analyzed using ImageJ (NIH, Bethesda, MD, USA).
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