The MitoTracker Deep Red FM probe (MitoTracker Mitochondrion-Selective Probes, Invitrogen European Headquarters, Paisley, PA4 9RF, UK) was used for mitochondrial staining, as previously described [7 (link)]. For immunofluorescence analysis, the cells were seeded and further processed, as reported by Iacopetta et al. [7 (link)]. The primary antibodies used were: rabbit anti-β-Actin and vimentin (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti cytochrome c and NF-κB p65 (Abcam, Cambridge, UK), diluted 1:100 in bovine serum albumin (BSA) 2% overnight at 4 °C. The secondary antibodies were: Alexa Fluor® 488 conjugate goat-anti-rabbit and Alexa Fluor® 568 conjugate goat-anti-mouse (Thermo Fisher Scientific, MA, USA), diluted 1:500 and incubated for 2h at 37 °C. DAPI 0.2 μg/mL (Sigma Aldrich, Milan, Italy) was used for nuclei staining. A fluorescence microscope (Leica DM 6000) was used for fluorescence detection. All the images were acquired and processed using the LAS-X software.
Alexa fluor 568 conjugate goat anti mouse
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor® 568 conjugate goat-anti-mouse is a fluorescent labeling reagent used for detecting and visualizing mouse antibodies in various research and diagnostic applications. It consists of a goat-derived anti-mouse antibody conjugated to the Alexa Fluor® 568 dye, which emits fluorescent signal in the orange-red spectrum when excited.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Dm6000, by Leica (4 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Primary mouse antibody, by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
N cadherin, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
5 protocols using alexa fluor 568 conjugate goat anti mouse
1
Mitochondrial and Immunofluorescence Staining
2
Immunofluorescence Analysis of NF-κB, iNOS, TNF-α, and COX
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Cells grown on glass coverslips in full medium were serum-deprived for 24 h and then treated with the complexes at a concentration of 25 µM for 24 h. After a PBS wash, cells were fixed using cold methanol (15 min at −20 °C) and incubated overnight at 4 °C with the primary antibodies raised against NF-κB, iNOS, TNF-α, and COX-1 and 2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in BSA 2%, as previously described [87 (link)]. Alexa Fluor®568 conjugate goat-anti-mouse (1:500 dilution), purchased from Thermo Fisher Scientific (Waltham, MA, USA), was used as the secondary antibody. DAPI (Sigma Aldrich, Milan, Italy) 0.2 µg/mL was added for 10 min for nuclei counterstaining. A fluorescence microscope (Leica DM 6000) was used for visualizing images, and LAS-X software 3.5.7.23225 was used for acquisition and processing. The fluorescence quantification was performed using Image J software 1.54d.
3
Immunocytochemical Analysis of Cell Markers
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For immunocytochemistry, cells were processed as already reported [28 (link),29 (link)]. The primary mouse antibody against E-cadherin, N-cadherin, vimentin, VEGF, actin and tubulin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The secondary antibodies Alexa Fluor® 568 conjugate goat anti-mouse was acquired from Thermo Fisher Scientific (Waltham, MA, USA). Nuclei were stained using DAPI 0.2 µg/mL (Sigma). Fluorescence was detected by using a fluorescence microscopy (Leica DM6000). All the images, processed with LAS-X software, are representative fields of three independent experiments.
4
Immunofluorescence Cytochrome C Localization
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Cells were grown on glass coverslips in full media, then serum-deprived for 24 h and exposed to compound to test, at the indicated time. Then, they were PBS-washed, fixed with cold methanol (15 min/−20 °C) and washed three times (10 min/room temperature) with cold PBS containing 0.01% TritonX-100. After incubation (30 min/room temperature) with blocking solution (PBS, 2% BSA), they were incubated with primary antibody diluted in blocking solution (4 °C/overnight). The mouse anti-cytochrome c (556433) was purchased from BD Biosciences (Franklin Lakes, NJ) and used at 1:100 dilution. Coverslips were then washed three times with PBS, then fixed cells were incubated with the secondary antibody Alexa Fluor® 568 conjugate goat-anti-mouse (1:500, Thermo Fisher Scientific, Waltham, MA). Nuclei were stained using DAPI (Sigma-Aldrich, Milan, Italy) for 10 min at a concentration of 0.2 µg/mL then washed three times with PBS. Fluorescence was detected using a fluorescence microscope (Leica DM 6000 Leica, Frankfurt am Main, Germany). LASX software was used to acquire and process all images42 .
5
Immunofluorescence Analysis of β-Actin
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Cells were seeded in 48‐well culture plates containing glass slides and then serum‐deprived for 24 h and incubated with the most active compound for 24 h (concentration equal to its IC50 value), as previously described.[40] The primary antibody used was mouse anti‐β‐actin (Santa Cruz Biotechnology, Dallas, USA) and the secondary antibody was Alexa Fluor® 568 conjugate goat‐anti‐mouse (Thermo Fisher Scientific, MA, USA). Fluorescence was detected using a fluorescence microscope (Leica DM 6000, 20x magnification). LAS−X software was used to acquire and process all images. Images are representative of three independent experiments.
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