NGFs and thereafter transformed CAFs were plated in six-well chamber slides (Nunc™, Thermo Fisher Scientific, Rochester, NY, USA) for 24 h. An immunofluorescence experiment was carried out using a previously established protocol according to the vendor’s instructions. Primary antibodies were then added and incubated at room temperature for 1 h. The primary antibodies used were α-smooth muscle actin (α-SMA, 1:100, cat no. 48938) and vimentin (Vim, cat no. 5741, 1:100, Cell Signaling Technology, Danvers, MA, USA). Matched secondary antibodies were anti-mouse immunoglobulin G (IgG) (H+L), F(ab)2 fragment (1:800, AlexaFluor 488 conjugate, cat no. 4408) and anti-rabbit IgG (1:600, AlexaFluor 555 conjugated, cat no. 4413). Stained cells were mounted using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) to counterstain the DNA. Cells were imaged on a Zeiss Axiophot (Carl Zeiss, Germany) fluorescence microscope. Microphotographs were captured using an AxioCam MRc digital video camera and analyzed using AxioVision Zeiss software (Carl Zeiss).
Vimentin vim
Manufactured by Cell Signaling Technology
Sourced in United States
Vimentin (VIM;) is a type III intermediate filament (IF) protein that is involved in maintaining the structural integrity of cells. It plays a crucial role in the organization and dynamics of the cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
H2s gas, by Airgas (2 mentions)
U plex combo kit against tnf α, by Mesoscale (2 mentions)
Primaryantibody against fas, by Santa Cruz Biotechnology (2 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (2 mentions)
Hif 1α, by Cell Signaling Technology (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rt2 sybr green rox qpcr mastermix, by Qiagen (2 mentions)
Axiophot, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Heat shock protein 70, by Abcam (1 mentions)
Bicinchoninic acid protein determination kit, by Thermo Fisher Scientific (1 mentions)
C casp3, by Cell Signaling Technology (1 mentions)
N cadherin n cad, by Cell Signaling Technology (1 mentions)
C parp, by Cell Signaling Technology (1 mentions)
β actin actb, by Cell Signaling Technology (1 mentions)
E cadherin e cad, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
8 protocols using vimentin vim
1
Immunofluorescence Analysis of CAFs
2
Protein Extraction and Western Blot Analysis from Diverse Biological Samples
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Proteins were extracted from MC38 cells, liver tissues, CRLM tissues, CRLM-adjacent tissues, and exosomes by a lysis buffer (50 mmol/L Tris, 1% NP40, 0.25% deoxycholic acid sodium salt, 150 mmol/L NaCl, 1 mmol/L EGTA), and protein concentrations were determined using a bicinchoninic acid protein determination kit (Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. Western blot analyses were performed with antibodies against SMPD3 (Santa Cruz Biotechnology), MMP2 (Cell Signaling Technology), cCASP3 (Cell Signaling Technology), cPARP (Cell Signaling Technology), BCL-2 (Affinity, Changzhou, Jiangsu, China), BAX (Cell Signaling Technology), cytochrome C (CYC) (Abcam), heat shock protein 90 (Abcam), heat shock protein 70 (Abcam), CD63 (Abcam), CD9 (Proteintech, Guangzhou, China), N-cadherin (NCAD) (Cell Signaling Technology), vimentin (VIM) (Cell Signaling Technology), E-cadherin (ECAD) (Cell Signaling Technology), CANTB (Cell Signaling Technology), CD31(Abcam), GAPDH (Abcam), Histong3 H3 (Abcam), and β-Actin (ACTB) (Cell Signaling Technology). ACTB was used as a loading control. The information on these primary antibodies is listed in Table 2 .
3
Genetically Engineered Mouse Models for Breast Cancer Research
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The generation of p18−/−, p18−/−;Brca1MGKO (p18−/−; Brca1f/f;MMTV-Cre or p18−/−;Brca1f/−;MMTV-Cre) and p16−/−;Brca1MGKO (p16−/−;Brca1f/f;MMTV-Cre or p16−/−; Brca1f/−;MMTV-Cre) mice has been described previously [23 (link), 37 (link), 39 (link)]. The Institutional Animal Care and Use Committee at the University of Miami approved all animal procedures. Histopathologic analysis and immunohistochemical analysis (IHC) were performed as described previously [19 (link), 23 (link), 37 (link)]. The primary antibodies used were Ck14 (Thermal Scientific), ERα (Santa Cruz), p-Akt (Ser473), p-4E-bp1 (Thr37/46), vimentin (Vim), E-cadherin (E-cad), p-Fra1 (Ser265) (Cell Signaling), Ki67 and fibronectin (Fn) (Abcam). Immunocomplexes were detected using the Vectastain ABC DAB kit according to the manufacturer’s instructions (Vector Laboratories) or by using Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Biolegend). The positive results of IHC were quantified by the H score, as previously described [41 (link)].
4
BDH2 Overexpression in Cell Lines
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We used the following antibodies: BDH2 (1:1000, HPA004428, Sigma-Aldrich, St. Louis, MO, USA), β-catenin (1:1000, sc-376841, Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin (1:1000, #3195P) and vimentin (VIM; 1:1000, #5741P, both Cell Signaling Technology, Beverly, USA), secreted protein acidic and cysteine rich (SPARC; 1:1000, #66426-1, SANYING, Wuhan, China) and GAPDH (1:10000, #5174P, Cell Signaling Technology). The secondary antibody 680RD goat anti-mouse and IRDye 800CW goat anti-rabbit antibodies were from LI-COR Biosciences (Lincoln, NE, USA).
BDH2 plasmid was purchased from Origene (Rockville, MD, USA); the open-reading frame of BDH2 was subcloned into the pCMV6-Entry vector. The protocol of transfection has been described.20 (link)
BDH2 plasmid was purchased from Origene (Rockville, MD, USA); the open-reading frame of BDH2 was subcloned into the pCMV6-Entry vector. The protocol of transfection has been described.20 (link)
5
Molecular Techniques for Hepatocyte Analysis
6
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Protein lysates were harvested from the cells using 1× RIPA lysis buffer (Nacalai Tesque, Kyoto, Japan) and kept on ice for 30 min before being centrifuged at 10,000 g for 10 min at 4 °C to obtain the supernatant. Protein lysates (50 μg) were subjected to SDS-polyacrylamide gel electrophoresis and western blot analysis as described [19 (link)]. Dilutions and the sources of the antibodies used are as follows: E-cadherin (CDH1, 1:1000, Cell Signaling Technology), occludin (OCLN, 1:1000, Merck Millipore), vimentin (VIM, 1:1000, Cell Signaling), SNAI1 (1:250, Cell Signaling) and GAPDH (1:2000, Cell Signaling). The protein bands were detected using a horseradish peroxidase-conjugated secondary antibody (1: 10,000, Abcam, Cambridge, UK) for 1 h at room temperature and visualized with the Amersham ECL Western blotting substrate (GE Healthcare), according to the manufacturer’s protocol. The mean of multiple blots is presented (Fig. 4b ); the error bars represent standard errors of the mean, SEM.
7
Molecular Techniques for Hepatocyte Analysis
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H2S gas was purchased from Airgas (La Porte, TX). RNeasy mini
kit was purchased from Qiagen (Germantown, MD). High Capacity cDNA RT kit were
purchased from ThermoFisher Scientific (Waltham, MA). RT2 SYBR Green
ROX qPCR Mastermix and primers for Gapdh were purchased from Qiagen (Valencia,
CA). Primary antibodies against Alb, hypoxia inducing factor 1 alpha
(Hif-1α), and Vimentin (Vim) were purchased from Cell signaling (Danvers,
MA). Primary antibodies against nuclear factor-like 2 (Nrf2) and 3-Oxoacid CoA
Transferase 1 (Oxctl) were purchased from Abeam (Cambridge, MA). Primary
antibody against Fas was purchased from SantaCruz Biotechnology (SantaCruz, CA).
Primary antibody against NeuN was purchased from Millipore (Billerica, MA).
U-PLEX combo kit against TNF-α was purchased from Meso Scale Diagnostics
(Rockville, MD).
kit was purchased from Qiagen (Germantown, MD). High Capacity cDNA RT kit were
purchased from ThermoFisher Scientific (Waltham, MA). RT2 SYBR Green
ROX qPCR Mastermix and primers for Gapdh were purchased from Qiagen (Valencia,
CA). Primary antibodies against Alb, hypoxia inducing factor 1 alpha
(Hif-1α), and Vimentin (Vim) were purchased from Cell signaling (Danvers,
MA). Primary antibodies against nuclear factor-like 2 (Nrf2) and 3-Oxoacid CoA
Transferase 1 (Oxctl) were purchased from Abeam (Cambridge, MA). Primary
antibody against Fas was purchased from SantaCruz Biotechnology (SantaCruz, CA).
Primary antibody against NeuN was purchased from Millipore (Billerica, MA).
U-PLEX combo kit against TNF-α was purchased from Meso Scale Diagnostics
(Rockville, MD).
8
Immunofluorescence Staining of Tumor Cells
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5,000-10,000 tumor cells were seeded on 8-well chamber slides (cat. no. 154534; Thermo Fisher Scientific, Inc.) and staining was performed according to the manufacturer's protocol using 5% donkey serum for blocking (cat. no. D9663¸ Sigma-Aldrich; Merck KgaA) and the following antibodies: Primary epithelial cell adhesion molecule (EpCAM, 1:200; cat. no. 2929; Cell Signaling Technology, Inc.) and vimentin (VIM; 1:200; cat. no. 5741; Cell Signaling Technology, Inc.) and secondary fluorescence-conjugated Cy™3 AffiniPure Donkey Anti-Mouse IgG (H+L; 1:500; cat. no. 715-165-150; Jackson ImmunoResearch) and fluorescein AffiniPure Donkey Anti-Rabbit IgG (H+L; 1:500; cat. no. 711-095-152; Jackson ImmunoResearch). Nuclei were stained using mounting medium with DAPI (cat. no. ab104139; Abcam). Fluorescence images (magnification, ×200) were taken with Axio Imager M2 microscope using ZEN 3.4.91.0 software (Carl Zeiss Microscopy GmbH).
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