RAW264.7 macrophages were washed with PBS, fixed with 4% paraformaldehyde and subsequently permeabilized using 0.3% Triton X-100. After blocking with 5% BSA, the cells were incubated with anti-LC3II (1:100, Proteintech Biotechnology, Chicago, USA), or anti-Arg-1 (1:100, Mouse IgG, Cell Signaling Technology, Boston, USA) and anti-iNOS (1:200, Rabbit IgG, Cell Signaling Technology, Boston, USA) antibodies overnight at 4 °C. Subsequently, cells were rinsed with PBS, then incubated with an FITC conjugated goat anti-rabbit secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) or Cy3 conjugated goat anti-mouse secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) and mounted with DAPI (1:200, Boster Biological Technology, China). Finally, the cells were photographed using a fluorescence microscope (Olympus Corporation, Japan).
Cy3 conjugated goat anti mouse secondary antibody
Manufactured by Wuhan Servicebio Technology
Sourced in China
The Cy3 conjugated goat anti-mouse secondary antibody is a lab equipment product designed to detect and visualize mouse primary antibodies in various immunoassays. It contains a fluorescent Cy3 dye conjugated to a goat-derived antibody that binds to mouse immunoglobulins.
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Lab products found in correlation
5 protocols using cy3 conjugated goat anti mouse secondary antibody
1
Immunofluorescence Staining of Macrophages
2
Aortic Plaque Characterization by Histology and Immunofluorescence
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Whole arteries, including the aortic arch, thoracic, and abdominal regions, were cut longitudinally, fixed, and then stained with Oil Red O for lipid measurement at the surface of the vascular wall. The aortic root was embedded in paraffin, and then the aortic root was cut into 5 μm slices from the aortic valve. Three consecutive slices were dewaxed with xylene, then hydrated with alcohol from low to high concentration, and the morphological analysis was carried out by hematoxylin-eosin and Movat five-color dyeing in references [21 (link)]. Moreover, Three consecutive slices were fixed, and fluorescent antibody nitric oxide synthase (iNOS) and mannose receptor 1(CD206) diluted with a certain staining titer were dripped, and the staining analysis was carried out according to the literature to evaluate the polarization level of macrophages in aortic plaque [22 (link)]. In short, the sections were incubated with anti-inducible iNOS and CD206 antibody at 4 °C overnight, then incubated with an FITC conjugated goat anti-rabbit secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) or Cy3 conjugated goat anti-mouse secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) and mounted with DAPI (1:200, Boster Biological Technology, China). Images were obtained using a confocal microscope and were merged using Image Pro Plus.
3
Quantification of Adenovirus Infection in A549 Cells
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The A549 cells grown in a 12-well or 96-well plate were infected with HAdV-55, rAd55, and rAd55-dE3-EGFP, respectively. The viral culture was 10-fold diluted in series. The infected cells were cultured for 40–48 h in culture at 37 °C and 5% CO2 and then we removed the medium. The cells were fixed with cold methanol at −20 °C for 10 min. After 30 min blocking with PBS containing 1% bovine serum albumin (BSA-PBS), the cells were incubated with hexon-specific antibody (B025/AD51, abcam, UK) at 37 °C for 1 h, then with a CY3-conjugated goat anti-mouse secondary antibody (Servicebio, Wuhan, China) for another 1 h. Then, 40, 60-diamidino-2-phenylindole (DAPI, Servicebio, China) was used for nuclear staining. The cells were observed and photographed under a fluorescence microscope at the excitation/emission wavelengths of 470 nm/515 nm, respectively.
4
Immunofluorescence Analysis of RAW264.7 Macrophages
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RAW264.7 macrophages were washed with PBS, xed with 4% paraformaldehyde and subsequently permeabilized using 0.3% Triton X-100. After blocking with 5% BSA, the cells were incubated with anti-LC3II (1:100, Proteintech Biotechnology, Chicago, USA), or anti-Arg-1 (1:100, Mouse IgG, Cell Signaling Technology, Boston, USA) and anti-iNOS (1:200, Rabbit IgG, Cell Signaling Technology, Boston, USA) antibodies overnight at 4 ℃. Subsequently, cells were rinsed with PBS, then incubated with an FITC conjugated goat anti-rabbit secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) or Cy3 conjugated goat anti-mouse secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) and mounted with DAPI (1:200, Boster Biological Technology, China). Finally, the cells were photographed using a uorescence microscope (Olympus Corporation, Japan).
5
Immunofluorescence Staining of Macrophages
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RAW264.7 macrophages were washed with PBS, fixed with 4% paraformaldehyde and subsequently permeabilized using 0.3% Triton X-100. After blocking with 5% BSA, the cells were incubated with anti-LC3II (1:100, Proteintech Biotechnology, Chicago, USA), or anti-Arg-1 (1:100, Mouse IgG, Cell Signaling Technology, Boston, USA) and anti-iNOS (1:200, Rabbit IgG, Cell Signaling Technology, Boston, USA) antibodies overnight at 4 . Subsequently, cells were rinsed with PBS, then incubated with an FITC conjugated goat anti-rabbit secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) or Cy3 conjugated goat anti-mouse secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) and mounted with DAPI (1:200, Boster Biological Technology, China). Finally, the cells were photographed using a fluorescence microscope (Olympus Corporation, Japan).
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