Approximately 1 × 104 astrocytes were seeded in 24-well plates. After 24 h of hypoxia, the pre-treated astrocytes were fixed with paraformaldehyde for 30 min, and then cells were permeabilized with 0.3% Triton X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for 1 h. Subsequently, cells were incubated with primary antibody against rabbit anti-Nrf2 (1:200, Abcam) at 4 °C, overnight. After washing with PBS for three times, cells were incubated with Alexa Fluor 555-conjugated donkey anti-rabbit secondary antibody (1:800, Invitrogen, CA, USA) at room temperature for 1 h. The cell nuclei were stained with DAPI. The images were obtained using a Leica microscope (×20). The assay was done in triplicate per separate experiment.
Alexa fluor 555 conjugated donkey anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555-conjugated donkey anti-rabbit secondary antibody is a laboratory reagent used for the detection and visualization of rabbit primary antibodies in various research applications. It consists of a donkey-derived secondary antibody labeled with the Alexa Fluor 555 fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Microscope 20, by Leica (1 mentions)
Ab52199, by Abcam (1 mentions)
Ab154610, by Abcam (1 mentions)
Ab18983, by Abcam (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Sudan black b, by Merck Group (1 mentions)
Aqua poly mount medium, by Polysciences (1 mentions)
Dm4000b fluorescence microscope, by Leica (1 mentions)
Hoechst 33342 nuclear stain, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
4 protocols using alexa fluor 555 conjugated donkey anti rabbit secondary antibody
1
Nrf2 Activation in Hypoxic Astrocytes
2
Immunofluorescent Localization of PLC Isoforms
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MA were cut longitudinally, and pinned (endothelium en face) on a Sylgard block. Arteries were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 then blocked with 4% normal donkey serum. Following overnight incubation at 4°C with primary antibodies (1:500; PLCβ3 Abcam #ab52199; PLCδ1 Abcam #ab154610; PLCγ2 Abcam #ab18983) in 0.1% Triton X-100, arteries were incubated for 1h with an Alexa Fluor 555-conjugated donkey anti-rabbit secondary antibody (Invitrogen). DAPI was applied to stain nuclei prior mounting for confocal imaging. Autofluorescence of the internal elastic lamina was also acquired. Fluorescence emissions were detected using a Zeiss LSM 510 confocal microscope (63X oil objective/1.4, ex: 405 nm, 488 nm and 543 nm). All images were deconvolved with Huygens professional software using experimentally determined point spread functions (PSFs) and reconstructed with Zen 2009 Light Edition.
3
Dual Immunofluorescence Imaging of p65 and CD31 in Mouse Kidney
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Immunofluorescence double staining was performed on 5-μm cryosections from snap-frozen mouse kidneys. After acetone fixation, sections were incubated with primary rabbit anti-p65 antibody (1:200, #D14E12; Cell Signaling Technology, Leiden, The Netherlands) and rat anti-CD31 antibody (1:200, #550274; BD Pharmingen, San Diego, Calif) in PBS containing 5% FCS for 1 h at RT. After washing, sections were incubated with Alexa Fluor555-conjugated donkey anti-rabbit secondary antibody (A-31572; Thermo Fisher, Waltham, Mass) to detect p65, and goat anti-rat Alexa Fluor488 secondary antibody to detect CD31 (A-11006; Thermo Fisher) in PBS with 5% FCS plus 2% normal mouse serum (Sanquin) for 45 min at RT. After washing, sections were incubated with 0.1% Sudan Black B (Sigma-Aldrich) in 70% ethanol for 30 min at RT. Thereafter, sections were washed 3 times and mounted in Aqua/Polymount medium containing DAPI (Polysciences, Warrington, Pa). Fluorescence images were taken using a Leica DM4000B fluorescence microscope (Leica Microsystems Ltd., Germany) with Leica LAS V4.5 image software. All images were taken with equal exposure times.
4
Quantifying GluN2A Membrane Localization
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48-hours after transfection HEK cells were treated in two different ways: 1) For total GluN2A labelling, cells were fixed with 4% paraformaldehyde for 10 min before blocking/permeabilisation with 5% BSA (Thermo Fisher) 0.1% Triton X-100 (Sigma) in PBS for 30 min at room temperature. 2) For membrane-only staining, cells were blocked in PBS with 5% BSA and 1mM Ketamine for 1 hour at room temperature. For both methods, HA-tagged GluN2A was immunolabelled using anti-HA antibody (H6908) at 5 µg/ml in PBS with 0.5% BSA, (and 0.1% Triton X-100 for the total GluN2A labelled cells), for 2 hours at 4 °C, followed by washing. The membrane-only cells were now fixed with 4% paraformaldehyde. Both cell types were then incubated for 1.5 hours with Alexa Fluor 555-conjugated donkey-anti-rabbit secondary antibody at 1:1000 in the dark (A-31572, Thermo Fisher). Cells were finally then incubated for 10 min at room temperature with Hoechst 33342 nuclear stain (Thermo Fisher). Stained cells were viewed by confocal microscopy using the Olympus FV1000. Image files were analysed by using Cell Profiler24 (link). Briefly images were segmented by using the nuclear staining channel followed by measurement of average fluorescence intensity in the Alexa-555 channel.
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