HRGECs were allowed to grow to confluence on fibronectin-coated glass chamber slides and exposed to Cd for 24 h. The monolayers of HRGECs were then washed with PBS containing 100 mM glycine, fixed with 4% paraformaldehyde for 5 min, and washed three times with PBS for 10 min. Immunoreactivity was examined by staining with a rabbit polyclonal anti NF-κB p65 antibody (1:400; Cell Signaling Technology) overnight at 4°C and incubation with an Alexa 546-labeled anti-rabbit secondary antibody (1:200; Molecular Probes, Eugene, OR, USA) for 2 h. Nuclear staining was achieved using DAPI (1:1,000; Molecular Probes) and images of the the samples were captured using an Olympus LCX100 Imaging system (Olympus Corp., Tokyo, Japan) with an excitation wavelength of 546 nm.
Alexa 546 labeled anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 546-labeled anti-rabbit secondary antibody is a reagent used in immunodetection techniques. It is designed to bind and detect rabbit primary antibodies, allowing for the visualization and localization of target proteins or molecules in biological samples. The Alexa Fluor 546 dye provides a bright, photostable signal that can be detected using appropriate fluorescence detection methods.
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2 protocols using alexa 546 labeled anti rabbit secondary antibody
1
Investigating NF-κB Activation in HRGECs
2
Immunofluorescence Staining of Neural Cells
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After fixation for 15 min with 4% paraformaldehyde at room temperature, the cells were permeabilized for 30 min in 0.1% Triton X100 with 5% goat serum diluted in phosphate-buffered saline (PBS). Then, the cells were incubated with 1% BSA, which was followed by overnight incubation with a primary antibody specific to MAP2 (1:200; Sigma-Aldrich, St. Louis, MO, USA) and γ-H2AX (1:400; Cell Signaling Technology, Beverly, MA, USA). After washing with PBS, the cells were incubated at room temperature for 1 h with Alexa 488-labeled anti-mouse secondary antibody (1:1000; Molecular Probes, Carlsbad CA, USA) and Alexa 546-labeled anti-rabbit secondary antibody (1:1000; Molecular Probes). Nuclei were stained with 2 μg/ml Hoechst 33342 (Sigma-Aldrich) for 15 min.
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