For Figure 1 , ANA staining patterns of TIN mAbs were visualized using an IIF kit (INOVA diagnostics). For subsequent studies, HEp-2 cells were fixed in methanol [17 (link)] and incubated with mAbs (50 μg/ml), followed by Alexa Fluor 488-conjugated goat anti-human IgG (2 μg/ml) and Hoechst 33342 (both Life Technologies). Selected slides were sequentially stained with anti-vimentin V9 (2 μg/ml, DAKO) or anti-giantin (1μg/ml, Abcam) followed by Alexa Fluor 568-conjugated donkey anti-mouse antibodies (2 μg/ml, Molecular Probes). Samples were imaged using a Leica SP5 II STED-CW laser scanning confocal microscope.
Anti vimentin v9
Manufactured by Agilent Technologies
Sourced in United States
Anti-vimentin (V9) is a mouse monoclonal antibody that recognizes the vimentin protein, which is a type III intermediate filament. Vimentin is often used as a marker for mesenchymal cells and is expressed in a variety of cell types, including fibroblasts, endothelial cells, and certain cancer cells. The V9 clone is a commonly used anti-vimentin antibody for immunohistochemical and immunocytochemical applications.
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Lab products found in correlation
Anti giantin, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Anti rabbit immunoglobulin g igg, by Cell Signaling Technology (1 mentions)
Anti β actin ac 15, by Merck Group (1 mentions)
Digital sight ds fi2, by Nikon (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Ab117866, by Abcam (1 mentions)
Anti e cadherin hecd 1, by Abcam (1 mentions)
Anti actin c4, by Merck Group (1 mentions)
Amersham ecl plus kit, by GE Healthcare (1 mentions)
Goat anti rabbit or anti mouse igg horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
4 protocols using anti vimentin v9
1
Immunofluorescence Staining and Imaging of Cells
2
Immunofluorescence Staining of Epithelial Markers
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Anti-zona occludens 1 (ZO-1; D7D12), anti-claudin-1 (D5H1D), anti-E-cadherin (24E10), anti-β-catenin (D10A8), anti-snail homolog 2 (slug; C19G7), and anti-rabbit immunoglobulin G (IgG) were from Cell Signaling Technology (Danvers, MA, USA). The other antibodies were obtained from various sources: anti-vimentin (V9; Dako, Carpinteria, CA, USA), anti-cytokeratin-19 (CK-19; HPA002465), and anti-β-actin (AC-15) from Sigma-Aldrich (St. Louis, MO, USA); anti-mouse IgG from GE Healthcare (Buckinghamshire, UK); and all secondary fluorescent antibodies from Invitrogen (Carlsbad, CA, USA).
3
Immunohistochemical Staining Protocol
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For the IHC protocol, a Novolink Max-Polymer detection system was used, according to the manufacturer’s instructions. The detailed protocol is described in Supplementary Figure S1 . Primary antibodies included anti-E-cadherin (4A2C7; 1:1000 in BSA 5%; Invitrogen, MA, USA), anti-P-cadherin (56/P-Cadherin; 1:150 in BSA 5%; BD Biosciences, NJ, USA), anti-β-catenin (CAT-5H10; 1:900 in BSA 5%; Invitrogen, MA, USA), and anti-vimentin (V9; 1:1000 in BSA 5%; Dako, Denmark). The negative control refers to cells that were not incubated with the primary antibody. After IHC protocol, each coverslip was removed from the 24-well plate using a small pair of broad-tipped forceps and rinsed in water, counterstained in hematoxylin for 30 s, washed again for 5 to 10 min, dehydrated (at increasing concentrations of ethanol), and diaphanized in xylene, and the slides were mounted using Entellan. Each slide was analyzed using a Nikon Eclipse E600 microscope coupled to a digital camera (Nikon Digital Sight DS-Fi2, Tokyo, Japan). Images were then captured using the Imaging Software NIS-Elements AR Version 4.30.0 (Nikon, Tokyo, Japan).
4
Western Blot Analysis of EMT Markers
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Cells were lysed in Laemmli buffer and stored at À70 °C. Protein concentrations were determined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Samples (20-30 lg of total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Hybond â P; GE Healthcare, Little Chalfont, UK). Membranes were blocked for 1 h at room temperature with TBST (20 mM Tris, 137 mM NaCl, 3.8 mM HCl, and 0.1% [v/v] Tween â 20) containing 5% (w/v) nonfat milk powder, and blots were probed with anti-ID1, anti-ID2 or anti-ID3 (Z-8, C-20, C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-cadherin (HECD-1; Abcam, Cambridge, UK), anti-N-cadherin (32; BD Biosciences, Franklin Lakes, NJ, USA), anti-vimentin (V9; Dako, Glostrup, Denmark), anti-Snail (ab117866;
Abcam, Cambridge, UK), anti-MMP-9 (ab35326; Abcam) or anti-actin (C4; EMD Millipore, Billerica, MA, USA) antibodies for 1 h. Membranes were washed and incubated with a secondary antibody (either goat antirabbit or anti-mouse IgG-horseradish peroxidase) (Santa Cruz Biotechnology), washed again and developed for enhanced chemiluminescence using the Amersham ECL-Plus kit according to the manufacturer's instructions (GE Healthcare).
Abcam, Cambridge, UK), anti-MMP-9 (ab35326; Abcam) or anti-actin (C4; EMD Millipore, Billerica, MA, USA) antibodies for 1 h. Membranes were washed and incubated with a secondary antibody (either goat antirabbit or anti-mouse IgG-horseradish peroxidase) (Santa Cruz Biotechnology), washed again and developed for enhanced chemiluminescence using the Amersham ECL-Plus kit according to the manufacturer's instructions (GE Healthcare).
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