RIPA buffer (50 mM Tris-HCl, pH 7.5, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, 150 mM NaCl supplemented with complete proteinase inhibitor) was used to extract the whole protein from the neointimal hyperplasia, AAA and atherosclerotic tissues of mice. To ensure the same amount of proteins for each sample, supernatant was quantified by Bradford protein assay. Equal amounts of protein (30~60 μg) were separated by 10% SDS-PAGE, and electro-transferred to a PVDF membrane. Membranes were blocked with 5% non-fat milk in tris-buffered saline-Tween for 1 h at room temperature, and incubated with primary antibodies against SM22α (Abcam, ab14106, 1:1000), α-SMA (Abcam, ab5694, 1:1000), OPN (Proteintech, 22952-1-AP, 1:500) and β-actin (Santa Cruz, sc-47778, 1:1000) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (Abcam, ab205719, 1:20000) or anti-rabbit IgG (Abcam, ab205718, 1:20000) for 1 h at room temperature. The blots were evaluated with GE Image Quant™ LAS 4000 detection system. The protein bands of interest were quantified using Image Pro Plus 6.0 software, and the integrated signal densities were normalized to β-actin (the loading control).
Ab5694
Manufactured by Proteintech
Sourced in United Kingdom, United States
Ab5694 is a laboratory antibody product. It is a primary antibody that can be used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ab14106, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 detection system, by GE Healthcare (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Fluoviem fv 1000, by Olympus (1 mentions)
Anti cd63 antibody, by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Proteintech (1 mentions)
Anti tsg101 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti aldh1a1 antibody, by Proteintech (1 mentions)
Anti oct4 antibody, by Proteintech (1 mentions)
Anti β catenin antibody, by Proteintech (1 mentions)
Anti cd81 antibody, by Proteintech (1 mentions)
Anti α tubulin, by Abcam (1 mentions)
Anti e cadherin antibody, by Proteintech (1 mentions)
Anti n cadherin antibody, by Proteintech (1 mentions)
Ab2462, by Abcam (1 mentions)
A21203, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
7 protocols using ab5694
1
Western Blot Analysis of Neointimal, AAA, and Atherosclerotic Tissues
2
Immunocytochemistry of Cytoskeletal Markers
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Cells were firstly fixed using 4% paraformaldehyde, washed twice with PBS, and permeabilized with 0.2% TritonX-100. After blocking, incubating cells with primary antibodies specific for α-SMA (Abcam, ab5694), Vimentin (Proteintech, 10366-1-AP), or E-cadherin antibody (Proteintech, 20874-1-AP), overnight at 4 °C. After washing, cells were incubated with fluor-conjugated secondary antibodies (Invitrogen, A-11001) for 1 h, and DAPI for 5 min at room temperature. We observed the results with a laser scanning confocal microscopy.
3
Immunofluorescence and Immunohistochemical Analysis of Fibrotic Liver
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The primary LSEC from fibrotic mice were seeded on confocal dishes. Cells were fixed with 4% paraformaldehyde at 4 °C for 10 min. The livers were fixed with 4% paraformaldehyde at 4 °C for 6 h, dehydrated with 30% sucrose or gradient ethanol, embedded with optimal cutting temperature compound or paraffin, and sectioned into 10-μm-thick cross sections. For immunofluorescence or immunohistochemical assay, the samples were washed with PBS, then incubated with 0.3% triton-100 at 37 °C for 10 min, and incubated with PBS containing 1% BSA at 37 °C for 30 min. In addition, hydrogen peroxide was used to eliminate endogenous peroxidase. Next, the samples were incubated with primary antibodies, CXCR7 (1:200, Proteintech, 20423–1-AP, CHN), Stabilin2 (1:200, MBL, D317-3, JPN), α-SMA (1:200, Abcam, ab5694, UK), IL-1β (1:100, Proteintech, 26048-1-AP, CHN) overnight at 4 °C followed by fluorescent secondary antibodies or horseradish peroxidase (HRP) secondary antibodies to develop color. Images were captured using a laser scanning confocal microscope (Olympus FluoViem FV 1000, Tokyo, JPN) or a phase microscope, and signals were analyzed quantitatively using Image J software.
4
Western Blot Analysis of Cellular Markers
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Protein extraction and western blotting were performed as described previously [46 (link)]. The antibodies were as follows: anti-α-SMA (abcam, ab5694), Vimentin (Proteintech, 10366-1-AP), anti-E-cadherin antibody (proteintech, 20874-1-AP), anti-CD63 antibody (santa cruze, sc-5275), anti-CD81 antibody (proteintech, 27855-1-AP), anti-TSG101 antibody (Santa Cruz, California, United States, sc-7964), anti-RNF43 antibody (bioss, bs-24331R), anti-β-catenin antibody (proteintech, 51067-2-AP), anti-ALDH1A1 antibody (proteintech, 15910-1-AP), anti-OCT4 antibody (proteintech, 11263-1-AP), anti-N-cadherin antibody (proteintech, 66219-1-Ig), and anti-α-tubulin (abcam, ab7291). Protein levels were normalized to α-tubulin and analyzed by Image J software.
5
Immunofluorescence Staining of Rat Brain
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After rat brain was fixed in paraformaldehyde, the paraffin section was made as previously described.25 Then, the paraffin section was baked for 15 min. Treat the sections with xylene and alcohol. Antigen retrieval: use citrate buffer solution (pH 6.0) repair sections for 10 min with high pressure, then natural cooling to room temperature. Wash sections by PBS for 3 times × 5 min. Transmembrane by heat: penetrate section by 3% TritionX‐100 for 30 min and wash section by PBS for 3 times for 5 min. Block section by 3% BSA for 30 min. Wash section by PBS for 3 times × 5 min. Incubate with primary antibody: dilute the primary antibody by antibody diluent and incubated overnight at 4°C. Wash section by PBS for 3 times × 5 min. Incubate with fluorescent second antibody: dilute antibody by PBS at 1:300 and incubate at 37°C for 30 min in dark. Wash section by PBS for 3 times × 5 min. Stain section by DAPI at room temperature for 2 min in dark. Wash section by PBS for 3 times × 5 min. Seal section by glycerol. Photographs were taken using Olympus DP80 fluorescence microscope. The primary antibodies for the following proteins were used: p‐EGFR (Cell Signaling, 3777), MMP‐2 (Abcam, ab2462), α‐SMA (Abcam, ab5694), and TNF‐α (Proteintech, 17590‐1‐AP). The fluorescent second antibodies (Invitrogen, A21206 and A21203) were used.
6
Comprehensive Characterization of the Tumor Microenvironment in PDAC
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H&E, IHC, and IF staining were performed in PDAC tissues from archived formalin-fixed paraffin-embedded (FFPE) tumors and were performed according to standard protocols. For IHC, the slides were incubated with PGP9.5 (Servicebio, GB11159, 1:500), p75NRT (Abcam, ab52987, 1:100), GFAP (Proteintech, 16825, 1:200), S100β (Servicebio, GB11359, 1:150), IL-6 (Abcam, ab9324, 1:50), α-SMA (Abcam, ab5694, 1:200), N-Cadherin (Proteintech, 22018-1-AP, 1:100), Fibronectin (Proteintech, 15613-1-ap, 1:200), FAP (Cell signaling technology [CST], 66562,1:100) followed by HRP conjugated Goat Anti-Mouse IgG (H + L) (Servicebio, GB23301, 1:200) or HRP conjugated Goat Anti-Rabbit IgG (H + L) (Servicebio, GB23303, 1:200) and diaminobenzidine (Brown) (DAB, Dako). The slides were counterstained with hematoxylin (Dako), which stains nuclei blue, contrasting with the brown of HRP-DAB. Fibroblast morphology was confirmed based on the two pathologists’ assessments. IF was performed according to the manufacturer’s instructions (Panovue, 10004100100). Briefly, slides were incubated with primary antibodies for S100β (Servicebio, GB11359, 1:500), p75NRT (Abcam, ab52987, 1:100), α-SMA (Abcam, ab5694, 1:200), IL-6 (Abcam, ab233706, 1:50), and IL-1α (Proteintech, 16765-1-AP, 1:50) overnight at 4 °C, and stained with HRP-labeled goat anti-mouse/rabbit IgG secondary antibodies.
7
Comprehensive Antibody Validation Protocol
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Primary antibodies used in our study were anti-βactin (ABSci, #AB21800, 1:3000), anti-GAPDH (Proteintech, 10494-1-AP, 1:8000-10000), anti-BMP4 (Abcam, ab39973, 1:1000), anti-BMP2 (Abcam, ab14933, 1:1000), anti-Runx2 (Abcam, ab23981, 1:1000), anti-α-SMA (Abcam, ab5694, 1:2000), anti-osteopontin (OPN) (Proteintech, 22952-1-AP, 1:2000), anti-caspase3 (CST, #9662, 1:200), anti-cleaved-caspase3 (CST, #9661, 1:200), anti-Bcl-2 (Proteintech, 12789-1-AP, 1:500), and anti-Bax (Proteintech, 50599-2-Ig, 1:1000). And the secondary antibodies were HRP-linked goat anti-rabbit antibody (Multiscience, GAR0072, 1:5000-10000) and HRP-linked anti-mouse antibody (CST, #7076, 1:3000-5000).
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