Samples were washed with PBS and fixed with 4% formaldehyde (Sigma Aldrich) for 1 h at room temperature. After embedding in a paraffin block, 8-μm-thick sections were cut and stained with Meyer’s hematoxylin and eosin Y solutions (Muto Pure Chemicals, Tokyo, Japan). For immunohistochemical staining, the sections were fixed in 10% formaldehyde (pH 7) overnight at room temperature. The samples were first blocked in PBS containing 1% bovine serum albumin (Thermo Fisher Scientific) and 0.01% Triton X-100 (Thermo Fisher Scientific) for 1 h at room temperature and subsequently incubated overnight with anti-vimentin (rabbit, primary antibody, Merck Millipore, Billerica, MA, USA) and anti-cytokeratin 15 (rabbit, primary antibody, Abcam, Cambridge, UK) at 4 °C. The sections were incubated with the corresponding secondary antibodies (highly cross-absorbed goat anti-IgG (H + L) Alexa Fluor 488 or goat anti-IgG (H + L) Alexa Fluor 555, Thermo Fisher Scientific) in blocking solution for 3 h at room temperature, and finally stained with DAPI (Sigma Aldrich) in PBS for 8 min. A confocal microscope (LSM 700; Carl Zeiss) was used for fluorescence imaging.
Anti vimentin
Manufactured by Merck Group
Sourced in United States, United Kingdom, Italy, Germany, China
Anti-vimentin is a laboratory reagent used in immunohistochemistry and Western blotting applications. It is a monoclonal antibody that specifically binds to the vimentin protein, which is commonly used as a marker for mesenchymal cells and certain types of cancer cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti e cadherin, by BD (14 mentions)
Anti β actin, by Merck Group (12 mentions)
Anti e cadherin, by Cell Signaling Technology (10 mentions)
Anti gapdh, by Santa Cruz Biotechnology (8 mentions)
Anti snail, by Cell Signaling Technology (7 mentions)
Anti n cadherin, by BD (7 mentions)
Anti fibronectin, by Merck Group (6 mentions)
Anti e cadherin, by Merck Group (6 mentions)
Anti β actin, by Santa Cruz Biotechnology (6 mentions)
Anti n cadherin, by Merck Group (5 mentions)
Anti gapdh, by Merck Group (5 mentions)
Anti ki67, by Abcam (4 mentions)
Anti slug, by Cell Signaling Technology (4 mentions)
Anti α sma, by Merck Group (4 mentions)
Anti e cadherin, by Abcam (4 mentions)
Anti erk1 2, by Cell Signaling Technology (4 mentions)
Anti gfap, by Agilent Technologies (3 mentions)
Anti bcl 2, by Cell Signaling Technology (3 mentions)
Anti p21, by Santa Cruz Biotechnology (3 mentions)
Anti flag, by Merck Group (3 mentions)
94 protocols using anti vimentin
1
Immunohistochemical Analysis of Cell Markers
2
Brain Histological Processing and Imaging
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Brain samples obtained via surgical resection were immediately fixed in 10% buffered formaldehyde for 24 h at room temperature, and the surgical specimens were processed and paraffin-embedded. All specimens were cut into 5 µm sections with a microtome (Leica Microsystems GmbH, Wetzlar, Germany) and stained with hematoxylin and eosin, and additional slides were submitted to automated 3,3-diaminobenzidine (DAB; Dako Autostainer Link 48; Agilent Technologies, Inc., Santa Clara, CA, USA) immunohistochemical staining using 5% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% bovine serum albumin (BSA; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and 0,2% of Triton X-100 (Sigma-Aldrich; Merck Millipore) with blocking buffer for 1 h at room temperature for anti-NeuN antibody (cat. no. ABN91; Merck Millipore) and anti-vimentin (cat. no. GA630, Dako, Glostrup, Denmark) diluted in blocking buffer (1:100). All reactions included positive and negative external control samples on the same slide. The slides were reviewed under a Zeiss Axiokop 40 microscope (Carl Zeiss Group,). All images were documented in TIFF uncompressed format with a Retiga 2000R color video camera (QImaging, Surrey, Canada).
3
Immunofluorescence Staining of Cytoskeletal Proteins
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Cells were grown on glass coverslips, fixed in cold methanol for 20 min at room temperature, permeabilised in 0.1% Triton X-100 (Fisher Scientific, Hampton, NH), blocked with 3% bovine serum albumin (BSA) in TTBS for 1 h at room temperature and then incubated with either anti-vimentin (V6389, Millipore Sigma, St. Louis, MO) diluted 1:250 in 3% BSA/TTBS or anti-pan-keratin (MA5–13156, ThermoFisher, Waltham, MA) diluted 1:250 in 3% BSA/TTBS overnight at 4 °C. As a negative control, samples were incubated with the equivalent concentration of normal mouse IgG. The coverslips were washed 3× in TTBS and incubated with goat anti-mouse IgG Alexa Fluor 488-conjugated secondary antibody (ThermoFisher, Waltham, MA) diluted 1:500 with 3% BSA/TTBS in the dark for 1 h at room temperature. The samples were subsequently stained with DAPI for 20 min and washed, and then coverslips were mounted using Vectashield (Vector Labs, Burlingame, CA) and visualised by fluorescence microscopy. As a negative control, coverslips were processed with normal mouse IgG at the equivalent concentration as the antibodies used.
4
Immunofluorescent Labeling of SMOC2 and Vimentin
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Following antigen retrieval in citrate solution at pH6, sections were labeled with anti-SMOC2 (1:100; R&D Systems, USA) and anti-Vimentin 1:1000 (Millipore-Sigma). Slides were subsequently exposed to donkey anti-mouse Cy3-conjugated secondary antibody (1:200; Jackson ImmunoResearch Laboratories, USA) and donkey antirabbit AF647-conjugated (1:200 Jackson ImmunoResearch Laboratories). Fluoroshield with DAPI (Millipore-Sigma) was used for nuclear staining and mounting. All images were analyzed through NIH ImageJ using a color threshold algorithm (identical threshold settings for compared image sets) written by Gabriel Landini (version v1.8) available at http://www.mecourse.com/landinig/software/software.html .
5
Immunohistochemical Profiling of Neural Stem Cells
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The following primary antibodies were used: anti-nestin (mouse, 1:1000, BD Pharmingen), anti-laminin (rabbit, 1:1000, Sigma), anti-Ki67 (rabbit, 1:100, Abcam), anti-vimentin (chicken, 1:1000, Millipore), anti-Sox2 (goat, 1:200, Santa Cruz), anti-DCX (goat, 1:100, Santa Cruz), anti-Tuj1 (mouse, 1:1000, Covance) and anti-heparan sulfate (mouse, 1:500, US Biological).
The following secondary antibodies were used: goat anti-mouse CY3 (Amersham), donkey anti-mouse 488 (Molecular Probes), goat anti-rabbit 488 (Molecular Probes), donkey anti-rabbit 488 (Molecular Probes), rabbit anti-chicken CY3 (Chemicon), donkey anti-goat 546 (Molecular Probe). Nuclei were stained with the nuclear marker TO-PRO3 (Invitrogen).
The following secondary antibodies were used: goat anti-mouse CY3 (Amersham), donkey anti-mouse 488 (Molecular Probes), goat anti-rabbit 488 (Molecular Probes), donkey anti-rabbit 488 (Molecular Probes), rabbit anti-chicken CY3 (Chemicon), donkey anti-goat 546 (Molecular Probe). Nuclei were stained with the nuclear marker TO-PRO3 (Invitrogen).
6
Multimodal Imaging of Neuronal and Glial Markers
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Sections (50 μm-thick) were rinsed in Tris-HCl phosphate buffer and then co-incubated overnight at room temperature with lectin-diluted with 1% BSA in Tris-HCl phosphate buffer or with the following primary antibodies: anti-vimentin (1:200, Millipore, Darmstadt, Germany); anti-βIII-tubulin (1:1000, Promega) and FITC-conjugated isolectin B4 (1:10, Sigma, St. Louis, MO, USA). After the sections were rinsed, they were co-incubated for 2 h at room temperature with the following fluorophore-conjugated secondary antibodies: Cy5-conjugated chicken anti-IgG (1:200, Jackson ImmunoResearch, Baltimore Pike, West Grove, PA, USA) and DyLight 547-conjugated mouse anti-IgG (1:200, Jackson ImmunoResearch). The sections were also incubated in Hoechst 33258 (Sigma, St. Louis, MO, USA) as a nuclear stain. Multi-labeled images were obtained by confocal spectral microscopy (LSM 780, Carl Zeiss). Z-sectioning was performed at 1 μm intervals, and optical stacks of at least 30 images were used for the analysis. Digital three-dimensional (3D) reconstructions were created using Zeiss LSM software (ZEN, Carl Zeiss Microscopy GmbH, Aalen, Germany).
7
Quantitative Immunoblotting of Signaling Proteins
8
Exosomal Protein Analysis in Prostate Cancer Cells
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PCa cells and exosomal lysates were prepared using RIPA buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and total proteins were quantified using bicinchoninic acid (BCA) assay reagents (Thermo-Scientific, Rockford, IL, USA). About 10–20 μg protein lysates were fractionated on 4–20% SDS-PAGE. Proteins were then transferred into nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), blocked in 5% BSA for 1h at room temperature. Membranes were incubated overnight at 4 °C with anti-integrin α2 (ITGA2), anti-CD9, anti-CD63, anti-E-cadherin, and anti-calnexin (GeneTex, Irvine, CA, USA), anti-c-Myc, anti-phosphorylated and total FAK, anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Vimentin (Millipore), anti-pERK1/2 (Cell Signaling, Danvers, MA, USA) antibodies. Membranes were incubated with appropriate secondary antibody for 1 h at room temperature. The specific protein bands were developed using the Clarity TM Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The developed signals were visualized by Odyssey ® Fc Imager and C-Digit Blot Scanner (LI-COR, Lincoln, NE, USA) and the densitometric analysis was performed by the Image studio Lite (LI-COR, Lincoln, NE, USA).
9
Immunohistochemical Staining of Cell Markers
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Slides were prepared as described [28 (link)], with modifications. Sections were incubated with anti-dsRED (1:50; Clontech), anti-human progesterone receptor (1:50; DakoCytomation, Carpinteria, CA), anti-human estrogen receptor (clone 6 F11, ThermoFisher Scientific, Waltham, MA), anti-vimentin (1:100; Millipore), anti-bovine cytokeratin 8/18 (1:2000; Fitzgerald Industries, Acton, MA), or anti-Ki67 (clone Ab-4; 1:1000; ThermoFisher Scientific) in 5 % horse serum in PBS at 4 °C overnight and detected with NovaRED (Vector Laboratories) or DAB (Invitrogen).
10
Immunofluorescence Staining of Organoid Sections
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Organoids were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned by Stanford Human Pathology and Histology Service Center. Sections (4–5 μm) were deparaffinized and stained with hematoxylin and eosin (H&E). For immunofluorescence staining, the following antibodies were used. Primary antibodies: anti-E-cadherin (BD, 610181, 1:1000), anti-vimentin (Millipore, AB1620, 1:1000), and anti-CD3 (Dako, A0452, 1:100). All secondary antibodies were used at 1:500 and were: IgG (H + L)-Texas Red (goat anti-mouse; Thermo Fisher, # T-862, 1:1000), IgY (H + L)-AF488 (goat anti-chicken; Thermo Fisher, # A-11039, 1:1000), and IgG (H + L)-Cy5 (goat anti-rabbit; Thermo Fisher, # A10523, 1:1000). All images were captured on a Zeiss Axio-Imager Z1 with ApoTome attachment.
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