To analyze the localization of CRP in the liver tissues, frozen liver sections were treated with the Blocking Solution (DAKO, Glostrup, Produktionsvej, Denmark) at room temperature for 40 minutes, and rabbit anti-CRP (1:50, Santa Cruz) was applied to the sections at 4℃ overnight. Samples were washed with PBS, and incubated with Alexa Fluor 568 (1:100, Invitrogen)-conjugated secondary antibody at room temperature for 1 hour. To detect the co-localization of albumin and CRP in WB-F344 cells incubated with siRNA-CRP and LCA, WB-F344 cells were fixed with 100% methanol (Merck, NJ, USA). The cells were reacted with Blocking Solution (Dako) for 1 hour at room temperature and mouse antialbumin (1:50, Santa Cruz) and rabbit anti-CRP (1:50, Santa Cruz) at 4℃ overnight. After reaction, the cells were incubated with Alexa 488 and 568 (1:100, Invitrogen)-conjugated secondary antibody at room temperature for 1 hour. The slides were stained with 4’, 6-diamidino-2-phenylindole (DAPI). The images were observed with a fluorescence microscope (Nikon, Tokyo, Minato, Japan). All experiments were performed in triplicate.
Alexa 488 and 568
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 and 568 are fluorescent dyes commonly used in biological research. Alexa Fluor 488 is excited by blue light and emits green fluorescence, while Alexa Fluor 568 is excited by green light and emits orange-red fluorescence. These dyes are designed for labeling proteins, nucleic acids, or other biomolecules for detection and visualization in various applications, such as fluorescence microscopy, flow cytometry, and immunoassays.
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Lab products found in correlation
Dm4000 upright microscopy, by Leica (3 mentions)
Olig2, by R&D Systems (3 mentions)
780 laser scanning confocal imaging system, by Zeiss (2 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (2 mentions)
Anti nestin mouse clone 10c2, by Thermo Fisher Scientific (2 mentions)
Anti ki67 mouse clone b56, by BD (2 mentions)
Anti vimentin mouse clone rv202, by BD (2 mentions)
Dm il fluorescence microscope, by Leica (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Tom20, by Santa Cruz Biotechnology (2 mentions)
Phospho drp1s616, by Cell Signaling Technology (2 mentions)
Titan confocal, by Leica (2 mentions)
Blocking solution, by Agilent Technologies (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Rabbit anti crp, by Santa Cruz Biotechnology (1 mentions)
Mouse anti albumin, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Methanol, by Merck Group (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
9 protocols using alexa 488 and 568
1
Localization of C-Reactive Protein in Liver
2
Immunofluorescence Staining of Centrosomal Proteins
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The following primary antibodies were used: rabbit anti-centrin (Merck Millipore, MA, USA, 20H5) at 1:100, mouse anti-acetylated tubulin (Sigma, MO, USA, T7451) at 1:1000, rabbit anti-Cep63 (Merck Millipore, MA, USA, 06-1292) at 1:100, rabbit anti-Pericentrin (Abcam, Cambridge, UK, ab448) at 1:500, mouse anti-protamine (Novus Biologicals, CO, USA, H00005619) at 1:100, rabbit anti-β-tubulin (Abcam, Cambridge, UK, ab6046) at 1:1000, mouse anti-α-tubulin (Sigma, MO, USA, DM1A T6199) at 1:1000 in sperm and at 1:100 in oocytes and parthenotes, mouse anti-γ-tubulin (Sigma, MO, USA, GTU-448) at 1:100, rabbit anti-Poc1B (Thermo Fisher Scientific, MA, USA, PA5-24495) at 1:100, rabbit anti-WDR62 (Bethyl Laboratories, TX, USA, A301-560A) at 1:100, rabbit anti-Nek9 (Dr. Joan Roig gift) at 1:100, rabbit anti-Pontin (Home Made) at 1:100 and rabbit anti-Reptin (Home Made) at 1:100. Secondary antibodies anti-rabbit and anti-mouse conjugated to Alexa-488 and 568 (Invitrogen, CA, USA) were used at 1:1000 in sperm and cell culture, and 1:100 in oocyte and parthenotes for IF and 680 (Invitrogen, CA, USA) or IRdye 800 CW (LI-COR Biosciences, NE, USA) at 1:10 000 for western blots. Hoechst 33342 (1 µg/ml, Invitrogen, CA, USA) was used to visualize DNA.
3
Immunofluorescence Staining of Cell Cultures
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Cell cultures were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min, washed, and permeabilized with PBS containing 0.1% Triton X-100 (Fisher Scientific) and 4% normal goat serum (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 20 min, followed by the incubation of primary antibodies overnight at 4 °C. After three 10 min PBS washes; the cells were incubated with secondary antibodies for 1 h at room temperature followed by extensive washes. The antibodies included: anti-Nestin (mouse clone 10C2, 1:100, eBioscience), anti-Ki67 (mouse clone B56, 1:100, BD Biosciences), anti-glial fibrillary acidic protein (GFAP, mouse clone GA5, 1:500, eBioscience), anti-Vimentin (mouse clone RV202, 1:200, BD Bioscience), anti-alpha smooth muscle actin (SmA, rabbit clone E184, 1:100, Abcam, Cambridge, MA USA), and anti-PECAM (rabbit, 1:100, Abcam). Goat anti-mouse or rabbit Alexa 488 and 568 (1:250; Invitrogen) secondary antibodies were used. Fluorescence images were acquired on a Leica DM IL fluorescence microscope using an excitation/emission (Ex/Em) of 470/525 nm for Alexa 488 and Ex/Em of 560/645 nm for Alexa 568. Confocal images were acquired on a Zeiss 780 laser scanning confocal imaging system.
4
Immunocytochemical Characterization of Cell Cultures
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Cell cultures were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 20 min, washed, permeabilized with PBS containing 0.1% Triton X-100 (Fisher Scientific) and 4% normal goat serum (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 20 min, followed with incubation of primary antibodies overnight at 4°C. After three 10 min PBS washes, cells were incubated with secondary antibodies for 1 hr at room temperature, followed by three washes. Antibodies included: anti-βIII-tubulin (TUJ1, mouse clone 2G10, 1:500, eBioscience; rabbit, Sigma), anti-glial fibrillary acidic protein (GFAP, mouse clone GA5, 1:500, eBioscience; rabbit, Thermo Fisher), anti-Nestin (mouse clone 10C2, 1:100, eBioscience), anti-Ki67 (mouse clone B56, 1:100, BD Biosciences), anti-Vimentin (mouse clone RV202, 1:200, BD Bioscience). Goat anti-mouse or rabbit Alexa 488 and 568 (1:250; Invitrogen) secondary antibodies were used. Fluorescence images were acquired on a Leica DM IL fluorescence microscope using excitation/emission (Ex/Em) of 470/525 nm for Alexa 488, and Ex/Em of 560/645 nm for Alexa 568. Confocal images were acquired on a Zeiss 780 laser scanning confocal imaging system.
5
Imaging Xenografted Brain Tissue
6
Imaging Xenografted Brain Tissue
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Cells or 10 µm thick slices of xenografted brain tissue were fixed in 4% paraformaldehyde and immunolabeled using the following antibodies: DRP1 (BD Biosystems), phospho-DRP1S616 (Cell Signaling, Danvers, MA), TOM20 (Santa Cruz Biotechnology, Santa Cruz, CA), OLIG2 (R&D Systems), and SOX2 (R&D Systems). Primary antibodies were incubated overnight at 4°C, followed by species appropriate secondary antibodies (Alexa 488 and 568; Invitrogen Molecular Probes, Eugene, OR) with incubation for 1 hour. Nuclei were stained with DAPI, and slides were then mounted using Fluoromount (Calbiochem, San Diego, CA). Images were taken using a Leica Titan confocal or DM4000 Upright microscopy.
7
Immunofluorescence Staining of Cultured Cells
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Cultured cells, fibers, and muscle sections were fixed with acetone or 4% formaldehyde solution and blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 5% goat serum. After blocking, they were stained with the following primary antibodies: anti-MyHC (clone MF20; eBioscience), cleaved caspase-3 (clone 5A1E; Cell Signaling Technology, Danvers, MA, USA), antilaminin-a2 (clone 4H8-2; Sigma), and anti-GFP (EMD Millipore, Billerica, MA, USA). After staining, they were incubated with a secondary antibody conjugated with Alexa-488 and -568 (Molecular Probes). After primary and secondary staining, EdU staining was performed using the Click-iT EdU Imaging Kit (Invitrogen), according to the manufacturer’s instructions. Nuclei were stained with 4,6′-diamidino-2-phenylindole (DAPI). Stained cells or sections were analyzed using a BZ-X810 fluorescence microscope (Keyence, Osaka, Japan). For mitochondrial analysis, MBs were incubated for 1 h with the MitoTracker® Deep Red FM (#8778; Cell Signaling), a dye that stains the mitochondria in live cells. Stained cells were analyzed using a confocal laser scanning microscope (SPF5; Leica).
8
Immunostaining of Xenografted Brain Tissue
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Cells or 10 μm thick slices of xenografted brain tissue were fixed in 4% paraformaldehyde and immunolabeled using the following antibodies: CDC20 (Santa Cruz Biotechnology, Santa Cruz, CA), OLIG2 (R&D Systems), and SOX2 (R&D Systems). Primary antibodies were incubated overnight at 4°C, followed by species appropriate secondary antibodies (Alexa 488 and 568; Invitrogen Molecular Probes, Eugene, OR) with incubation for 1 hour. Nuclei were stained with DAPI, and slides were then mounted using Fluoromount (Calbiochem, San Diego, CA). Images were taken using a Leica DM4000 Upright microscopy.
9
Drosophila Larval Neuromuscular Junction Immunostaining
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