Following treatment with 2 μg/mL MIF in the presence or absence of 50 μM 4-IPP for 24 h, primary joint capsule fibroblasts were fixed in 4% paraformaldehyde with 0.1% Triton X-100 (Sigma, St Louis, MO, USA) and subsequently incubated with 4% goat serum to block nonspecific binding. Then, cells were incubated with anti-TGF-β1 (Servicebio, Wuhan, China, 1:100), anti-MIF (Abcam, 1:100), or anti-CD74 (Santa Cruz, 1:50) primary antibodies overnight. Fluorescent secondary antibodies of FITC-labeled goat anti-rabbit IgG (Sigma, 1:400) and Cy3-labeled goat anti-rabbit IgG (Sigma, 1:400) were used to visualize the corresponding subsets. Cells were then stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma, 1:4000) and phalloidin (Abcam, 1:1000), followed by observation under a confocal fluorescence microscope (Leica, Germany) and semi-quantitatively analyzed with Image-J software (National Institutes of Health, USA). The fluorescence was scored and quantified with blinding.
Cy3 labeled goat anti rabbit igg
Manufactured by Merck Group
Cy3-labeled goat anti-rabbit IgG is a laboratory reagent used in immunoassays and related techniques. It consists of goat-derived antibodies specific to rabbit immunoglobulin G (IgG), which have been labeled with the fluorescent dye Cy3. This product can be used to detect and visualize the presence of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Triton x 100, by Merck Group (1 mentions)
Confocal fluorescence microscope, by Leica (1 mentions)
Fitc labeled goat anti rabbit igg, by Merck Group (1 mentions)
Anti cd74, by Santa Cruz Biotechnology (1 mentions)
Anti tgf β1, by Wuhan Servicebio Technology (1 mentions)
Anti mif, by Abcam (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
Anti e cadherin antibody, by Proteintech (1 mentions)
Anti vimentin antibody, by Proteintech (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Vimentin, by Abcam (1 mentions)
Tgf β1, by Wuhan Servicebio Technology (1 mentions)
3 protocols using cy3 labeled goat anti rabbit igg
1
Immunofluorescence Analysis of Joint Fibroblast Responses
2
Immunofluorescence Assay of Ishikawa Cells
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Different groups of Ishikawa cells cultured in chamber slides were fixed in 4% paraformaldehyde, followed by permeabilization and blocking. Cells were then incubated with respective primary antibodies, including anti-vimentin antibody (1:2500, Proteintech, Hubei, China), anti-E-cadherin antibody (1:2000, Proteintech), overnight at 4°C. Cy3-labeled goat anti-rabbit IgG (1 μg/mL, Sigma) and goat anti-mouse IgG (1 μg/mL, Sigma) were used, respectively, to visualize the signal, and nuclei were stained with DAPI (1 μg/mL, Sigma). The cell pictures were observed under an inverted fluorescence microscopy imaging system (Olympus).
3
Immunohistochemical Analysis of Knee Joint Samples
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Knee joint samples were collected from experimental models at different time points, xed in 4% paraformaldehyde, decalci ed in 10% EDTA, and then embedded in para n. The knee joint specimens were sagittally sectioned (5 µm) and processed with hematoxylin-eosin (HE) and Masson trichrome staining. For immunohistochemical staining, the sections were co-incubated with antibodies against MIF (Abcam, 1:100), TGF-β1 (Servicebio, 1:100), and vimentin (Abcam, 1:1000). The sections were further incubated with FITC-labeled goat anti-mouse IgG (Gibco, 1:400), Cy3-labeled goat anti-rabbit IgG (Sigma, 1:400), and DAPI (Sigma, 1:4000). Zeiss LSM710 confocal microscope was used for confocal imaging of samples at an original magni cation × 40. Confocal images were prepared with Zen software. All images were taken from similar areas of the posterior joint capsule.Three sequential specimens in each group were measured.
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