THP-1 cells were seeded in a 24 well plate with coverslips and differentiated at 80% confluency. Cells were fixed with 2.5% formaldehyde for 20 min and the reaction stopped by adding 0.1 M glycine for 5 minutes (min). Cells were then permeabilized with 0.2% Triton X100 in PBS for 5 min. Cells were incubated for 1 h with primary antibody (rabbit-anti p65 mAB, Cell Signaling, cat. no. 8242S) 1:200 diluted in BSA/PBS 1 mg/ml. Cells were then washed three times with PBS and incubated in the dark for 45 min with Alexafluor594 secondary antibody (1:100) (Cell Signaling, cat.# 8889S) in BSA/PBS 1 mg/ml and incubated in the dark for 45 min. Cells were washed three times with PBS. To stain nuclei, DAPI was added at 1:10,000 in PBS for 10 min. Cells were washed three times with PBS and mounted with 20 μl of DAKO mounting medium per coverslip on glass slides and stored in the dark until imaged. The images were analysed using Image J s/w (imagej.net ).
Alexa fluor 594 secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 594 secondary antibody is a fluorescent dye-conjugated secondary antibody used for detection and visualization in various immunoassays and microscopy techniques. It is designed to bind and detect primary antibodies, allowing for signal amplification and increased sensitivity in the analysis of target proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (3 mentions)
Axiovert 135 microscope, by Zeiss (2 mentions)
Brdu labeling reagent, by Thermo Fisher Scientific (1 mentions)
Anti brdu primary antibody, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Alexa fluor 488 secondary antibody, by Cell Signaling Technology (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
6 protocols using alexa fluor 594 secondary antibody
1
Immunofluorescent Staining of p65 in THP-1 Cells
2
BrdU Incorporation Assay Protocol
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For the BrdU (5-bromo-2′-deoxyuridine) incorporation assay, cells were cultured with a BrdU-labeling reagent (Life Technologies), performed DNA hydrolysis with 1 M HCl, and stained with an anti-BrdU primary antibody (Cell Signaling) and Alexa Fluor® 594 secondary antibody according to the manufacturer’s instructions.
3
Caspase-3 Expression in Cells
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The expression of caspase-3 was assessed by immunocytochemistry. Cells slides were fixed in 4% formaldehyde for 10 min and pre-incubated in 0.5% Triton X-100 and 1.5% bovine serum albumin (BSA; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 15 min at room temperature. Immunostaining was performed by incubating overnight at 4°C with anti-caspase-3 (1:50, cat. no. 9662; Cell Signaling Technology, Danvers, MA, USA) followed by Alexa Fluor 594 secondary antibody (1:500, cat. no. R37117; Invitrogen, Thermo Fisher Scientific, Inc.) for 1 h at room temperature, washed twice with PBS. DAPI (5 g/l) was used for counterstaining for 5 min, and observed by a fluorescence microscope (Olympus Corp., Tokyo, Japan).
4
Quantifying Apoptosis and DUSP4 in Bovine Aortic Endothelial Cells
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Bovine aortic endothelial cells were cultured on sterile glass coverslips coated with attachment factor to an 80–90% confluence or 72 h post-transfection. Cells were subjected to H/R treatment (1 h hypoxia and 30 min reoxygenation), cell media was removed, and coverslips were washed 2× with PBS, fixed for 20 min at RT in 4% paraformaldehyde and subsequently washed 3× with PBS containing 0.1% BSA. Cells were then blocked for 45 min at RT in PBS containing 5% BSA and 0.3% TritonX-100 and then incubated overnight at 4°C in anti-cleaved caspase-3 or anti-DUSP4 diluted 1/400 in PBS with 1% BSA and 0.3% TritonX-100. After overnight incubation, coverslips were rinsed 3× with PBS containing 0.1% BSA, and incubated with Alexa Fluor 488 secondary antibody (green) for cleaved caspase-3 and Alexa Fluor 594 secondary antibody (red) for DUSP4 (Cell Signaling, Cambrdige, MA, USA) for 1 h at RT. Finally, coverslips were washed 3× with PBS containing 0.1% BSA, mounted onto a glass slide with ProLong Gold antifade reagent with DAPI (Life Technologies, Grand Island, NY, USA), and fluorescent images (40×) obtained using an Olympus FluoView FV1000 confocal microscope, and data were captured digitally and analyzed (18 (link), 21 (link)).
5
Cadmium-Induced Apoptosis Modulation
6
Immunofluorescence Assay for Apoptosis
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