HGFs were immersed in acetone and washed with PBS. Then, HGFs were fixed in 4% paraformaldehyde at 4°C overnight and washed with PBS three times. The following primary antibodies were used for cell incubation at 37°C for 2 h: Vimentin (1:100; ZM-0260; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and S100A4 (1:100; ZA-0257; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). H2O2 (3%) was added to blocking protease for 20 min at room temperature and then 30 µl sheep serum blocking buffer (ZC-02125; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) was added for 1 h at room temperature. Following three washes with PBS, the cells were incubated with the following secondary antibodies at 37°C for 1 h: Fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (IgG; 1:100, ZF-0312; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) and rhodamine-conjugated goat anti-rabbit IgG (1:100; ZF-0316; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole for 10 min at room temperature. Finally, the cells were photographed under a laser confocal microscope (Olympus Corporation, Tokyo, Japan).
Vimentin
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China
Vimentin is a type III intermediate filament protein that is widely expressed in various cell types, including mesenchymal cells. It is a key structural component of the cytoskeleton and plays a role in maintaining cell integrity and shape.
Automatically generated - may contain errors
Lab products found in correlation
Laser confocal microscope, by Olympus (1 mentions)
Zf 0312, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Zf 0316, by Zhongshan Golden Bridge Biotechnology (1 mentions)
2 step detection kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
E cadherin, by HuaAn Biotechnology (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Dab kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Phosphate buffered saline (pbs), by Zhongshan Golden Bridge Biotechnology (1 mentions)
Cytochrome c, by Abcam (1 mentions)
Ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Ezrin, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Beyotime (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Ab66218, by Abcam (1 mentions)
Ab113670, by Abcam (1 mentions)
D9542, by Merck Group (1 mentions)
5 protocols using vimentin
1
Immunofluorescence Analysis of Human Gingival Fibroblasts
2
Evaluating LLGL2 Expression in HCC Tissues
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Human HCC tissue microarrays (TMA) slides consisted of 156 pairs of primary tumor tissues and ANLT specimens. The immunohistochemical-staining assay for LLGL2(1:50), Vimentin(1:100), E-cadherin(1:100) was conducted using a 2-step detection kit (Zhongshan Golden Bridge Biotechnology, Beijing, China).Antibody Vimentin and E-cadherin purchased from HuaAn Biotechnology (Huaan, Hangzhou, China). We evaluated LLGL2 expression with an inverted microscope (Olympus, Japan). The levels of LLGL2 expression were scored on the basis of the percentage of the positively stained tumor cells. The grading score were; 4 indicates >80% positive, 3 indicates 51-80% positive, 2 indicates 31-50% positive, 1 indicates 5-30% positive, and 0 indicates ≤5% positive. The level of LLGL2 protein expression was classified into a high expression group (2 - 4) and a low expression group (0 - 1) for downstream analyses (33 (link)).
3
Immunohistochemistry Analysis of Rat Liver Tissue
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Diethylstilbestrol was obtained from Chengdu West Chemical Industry (China). Progesterone was purchased from Zhejiang Xian-Ju Pharmaceuticals (China). Absolute ethyl alcohol, xylene, aluminum potassium sulfate, sodium iodate, hydrochloric acid, potassium dichromate, and concentrated sulfuric acid were obtained from Chengdu Ke Long Chemical Industry (China). Paraformaldehyde was purchased from China National Group Corporation (China). PBS (0.01 M, pH 7.2–7.4), desmin, rabbit polyclonal antibody (1:200), α-SMA, rabbit polyclonal antibody (1:100), vimentin, goat anti-rabbit IgG, biotin (1:100), DAB kit, and citrate buffer solution (0.01 M, pH 6.0) were obtained from Beijing Zhong Shan Golden Bridge Biotechnology (China). Hematoxylin was obtained from Beijing J&K Scientific (China). Glycerin was purchased from Tianjin Fu Yu Fine Chemical Industry (China). Eosin was obtained from Tokyo Chemical Industry Co., Ltd (Japan) and neutral balsam from Shanghai Yi Yang Instruments (China).
4
Src Activation Regulates TSSC3 and RanBP9
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The primary antibodies used in this study were as follows: TSSC3 (Abnova, Taipei, Taiwan), RanBP9 (Abcam, Cambrige, MA, USA), cMyc (Santa Cruz Biotechnology, Dallas, TX, USA), Flag (Beyotime, Shanghai, China), ubiquitin (Santa Cruz Biotechnology), Src, Src Tyr418 (Cell Signaling Technology, Danvers, MA, USA), Akt, Akt Ser473, cleaved caspase-3 (Cell Signaling Technology), cytochrome c (Abcam), GAPDH (Cell Signaling Technology), Ezrin (Santa Cruz Biotechnology), BMP, vimentin and CK18 (Zhongshan Golden Bridge Biotechnology, Beijing, China).
Cells were treated with the Src activator pYEEI (Biomol Research Laboratories, Shanghai, China) for 12 h at a final concentration of 100 μmol/l and the Src inhibitor AZD0530 (Saracatinib;www.Selleckchem.com ) for 12 h at 3 μmol/l. Cycloheximide (Sigma, St. Louis, MO, USA) was applied for 24 h at a final concentration of 100 μg/ml.
Cytosolic proteins were extracted using the Mitochondria Isolation Kit for Mammalian Cells (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.
The primers used in this study were: RanBP9, 5′-CGCATCCAATACCAGCAGCC-3′ and 5′-GGCACAGTACCCATGGTGGA-3′ TSSC3, 5′-TCCAGCTATGGAAGAAGAAGC-3′ and 5′-GTGGTGACGATGGTGAAGTACA-3′ and GAPDH, 5′-CTTTGGTATCGTGGAAGGACTC-3′ and 5′-GTAGAGGCAGGGATGATGTTCT-3′.
Cells were treated with the Src activator pYEEI (Biomol Research Laboratories, Shanghai, China) for 12 h at a final concentration of 100 μmol/l and the Src inhibitor AZD0530 (Saracatinib;
Cytosolic proteins were extracted using the Mitochondria Isolation Kit for Mammalian Cells (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.
The primers used in this study were: RanBP9, 5′-CGCATCCAATACCAGCAGCC-3′ and 5′-GGCACAGTACCCATGGTGGA-3′ TSSC3, 5′-TCCAGCTATGGAAGAAGAAGC-3′ and 5′-GTGGTGACGATGGTGAAGTACA-3′ and GAPDH, 5′-CTTTGGTATCGTGGAAGGACTC-3′ and 5′-GTAGAGGCAGGGATGATGTTCT-3′.
5
Immunofluorescence Analysis of PDR Membranes
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IF staining was performed on 14 PDR membranes, which are seven samples from IVB treatment group, and seven samples from non-treatment group in 10 μm thick frozen sections. We used antibodies against the following antigens: TGFβ2 (1:200 dilution, ab113670, Abcam, Cambridge, Massachusetts, USA), CTGF (1:200 dilution, ab66218, Abcam), CNTF (1:200 dilution, ab66218, Abcam), VEGF (1:200 dilution, ab1316, Abcam) and vimentin (1:150 dilution, Zhongshan Golden Bridge Biotechnology, Beijing, China). Samples were counterstained with DAPI (1:1000 dilution, D9542, Sigma-Aldrich, USA). Sections were examined and photographed using a fluorescence microscope (DS-Ril-U2, Nikon, Tokyo, Japan).
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