At the end of the study, the tumor tissues were removed, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into 4 μm-thick cross sections. After deparaffinization, the paraffin sections were subjected to antigen retrieval with citrate buffer and treated with 3% H2O2 for 10 min at room temperature. The sections were then blocked with 5% non-immune goat serum for 30 min at 37°C and incubated with the corresponding primary antibodies at 4°C overnight. The primary antibodies used were rabbit anti-fascin antibody (1:1000, Abcam, ab126772) and rabbit anti-PTK6 antibody (1:1000, Abcam, ab233391). Horseradish peroxidase-conjugated secondary antibodies (PV-9001, ZSGB-Bio, Beijing, China) were added the next day for 30 min at 37°C, and a DAB kit (ZLI-9018, ZSGB-Bio) was used for color development according to the manufacturer’s instructions. Hematoxylin was used to counterstain the nuclei during IHC staining. Sections were incubated with non-immune IgG secondary antibody only, and no primary or secondary antibodies were used as negative controls.
Ab126772
Manufactured by Abcam
Sourced in United States
Ab126772 is a laboratory equipment product. It is a device used for scientific research and analysis. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab233391, by Abcam (2 mentions)
Pv 9001, by ZSGB-BIO (1 mentions)
Zli 9018, by ZSGB-BIO (1 mentions)
Dab kit, by ZSGB-BIO (1 mentions)
Aa128, by Beyotime (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ab32152, by Abcam (1 mentions)
Ab180714, by Abcam (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
A0216, by Beyotime (1 mentions)
Rabbit anti vegfr2, by Cell Signaling Technology (1 mentions)
Mouse anti cdc42, by BD (1 mentions)
Goat anti dll4, by R&D Systems (1 mentions)
Dylight 405 affinipure donkey anti goat igg h l, by Jackson ImmunoResearch (1 mentions)
Rabbit anti yap, by Cell Signaling Technology (1 mentions)
Ab8245, by Abcam (1 mentions)
A0208, by Beyotime (1 mentions)
Ab150077, by Abcam (1 mentions)
Chemiluminescent horseradish peroxidase substrate, by Merck Group (1 mentions)
Rabbit anti gapdh antibody, by Cell Signaling Technology (1 mentions)
10 protocols using ab126772
1
Immunohistochemical Analysis of Tumor Tissues
2
Quantifying Protein Expression by Western Blotting
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Western blotting was performed as previously described[30 (link)]. Primary antibodies specific to SYTL1 (1:500, PA5-24730, Thermo), FSCN1 (1:1000, ab126772, Abcam), ubiquitin (1:500, 10201, Proteintech, Wuhan, China), and GAPDH (1:1000, AA128, Beyotime, Shanghai, China) were used. The secondary antibodies were purchased from Cwbiotech (1:10,000, Beijing, China), and the blots were visualized using Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Merck Millipore, Germany).
3
Immunohistochemical Analysis of Tumor Samples
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Samples from the largest tumors were fixed in formalin for 24 h at room temperature, embedded in paraffin and sliced for hematoxylin and eosin staining. Immunohistochemistry was performed with primary Rab25 (ab106175, 1:200, Abcam, Cambridge, MA, USA), VEGFR-1 (ab32152, 1:250, Abcam), Snail (ab180714, 1:100, Abcam) and fascin (ab126772, 1:250, Abcam) antibodies, as described previously.25 (link) The investigator was blinded during the experiments.
4
Antibody Panel for Cellular Signaling
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The following primary antibodies were used: rabbit anti-FSCN1 (Abcam, ab126772; IF 1:200, WB 1:1000, IP 1:100), mouse anti-GAPDH (Abcam, ab8245; WB 1:1000), mouse anti-F-actin (Abcam, ab205; WB 1:1000), rabbit anti-YAP (Cell Signaling Technology, 14074; IF 1:200, WB 1:1000), rabbit anti-p-YAP (Ser127) (Cell Signaling Technology, 4911; WB 1:1000), mouse anti-CDC42 (BD Biosciences, 610929; WB 1:500), rabbit anti-VEGFR2 (Cell Signaling Technology, 2479; WB 1:1000), rabbit anti-p-VEGFR2 (Tyr1175) (Cell Signaling Technology, 2478; WB 1:1000), goat anti-DLL4 (R&D Systems, AF1389; IF 1:200), and mouse anti-β‐actin (Millipore, A5441; WB 1:1000). The following secondary antibodies were used: goat anti-mouse HRP-conjugated antibodies (Beyotime, A0216; 1:1000) and goat anti-rabbit HRP-conjugated antibodies (Beyotime, A0208; 1:1000) were applied for WB, Alexa Fluor™ Plus 488 Anti-mouse IgG (H + L) (Abcam, ab150077; IF 1:400), and DyLight 405-AffiniPure Donkey Anti-goat IgG (H + L) (Jackson ImmunoResearch,705–475-147,1:400) was applied for immunofluorescence.
5
Protein Expression Analysis Protocol
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Proteins were extracted from cells or fresh tissue lysates from the treatment and experimental groups. Protein extracts were prepared in RIPA lysis buffer containing PMSF (100:1). A BCA protein assay kit (Cwbiotech, Beijing, China) was used to determine the protein concentration. Protein extracts were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, MA, United States ), blocked with 5% BSA for 1 h at room temperature, and then incubated with the corresponding primary antibodies overnight. Finally, the proteins were visualized using chemiluminescent horseradish peroxidase substrate (Millipore). The levels of target proteins, such as β-actin, were normalized to those of the loading control. Six independent experiments were performed to derive the mean values for statistical analysis. The primary antibodies used were rabbit anti-fascin antibody (1:1000, Abcam, Cambridge, UK, ab126772), rabbit anti-PTK6 antibody (1:1000, Abcam, ab233391), and rabbit anti-GAPDH antibody (1:1000, Cell Signaling Technology, MA, United States , 2118).
6
Immunohistochemical Analysis of Murine Xenograft Tumors
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The murine xenograft tumor specimens were stained with antibodies against FSCN1 (Abcam; ab126772; 1:500), Ki-67 (Abcam; ab15580; 1:200) and Cleaved Caspase-3 (Abcam; ab2302; 1:20) and the staining intensity was measured in both groups. Immunohistochemical staining of 4 μm-thick TMA slides with ESCC and adjacent normal esophageal tissue samples was performed as previously described [47 (link)].
7
SYTL2 Regulates FSCN1 Ubiquitination
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PC3 cell lines, with or without stable overexpression of SYTL2, were seeded in 150 mm2 plates. Following adhesion, the cells were treated with MG-132 (10 µM), and cell lysates were harvested and incubated with anti-FSCN1 (ab126772, Abcam) as described for the Co-IP assay. The cell lysates eluted from the magnetic beads were immunoblotted with the anti-Ub antibody (1:500, 10201, Proteintech).
8
Comparing Human Liver Cancer Cell Lines
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Five human HCC cell lines (SNU387, Huh7, Hep3B, and SNU449) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Huh7 cells were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), Hep3B cells were cultured in minimum essential medium, and SNU387, SNU449 cells were incubated in RPMI-1640 complete medium (Gibco; Thermo Fisher Scientific, Inc.). All cell media were supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml streptomycin and penicillin. All cell lines were incubated at 37°C in 5% CO2. DOX and DAPI were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The following antibodies were used: FSCN-1 (1:1,000, ab126772), while antibodies against β-actin (1:1,000, ab8227), Twist (1:1,000, ab50581), E-cadherin (1:1,000, ab40772), Vimentin (1:1,000, ab8978) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (1:2,000, ab6721), all of which were purchased from Abcam (Cambridge, MA, USA), whereas HRP-conjugated goat anti-mouse secondary antibodies (1:2,000, 7076) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
9
Immunohistochemical Analysis of HCC Biomarkers
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The medical approval of this study was received from the Ethics Committee of The Second Affiliated Hospital of Xi’an Jiaotong University. IHC validation of key gene expressions was performed in tissues of HCC patients. Tissue samples were soaked in a dewaxing solution and 95% ethanol for dewaxing, then heated in the microwave to recover the antigen. After being blocked with 5% goat serum, Anti-FAM110D (ab234868, 1:500, Abcam, USA), anti-FBLN5 (ab66339, 1:500, Abcam), anti-FSCN1 (ab126772, 1:500, Abcam), Anti-pcdh17 (ab128815, 1:500, Abcam) Anti-PDE2A (ab224616, 1:500, Abcam) Anti-PGF (10642-1-AP, 1:500, Proteintech, China) were used for IHC analysis. Tissue samples were incubated with antibodies at 4°C for 8 h and then co-incubated secondary antibodies at room temperature for 60 min. The immunoreactivity was observed after incubation with diaminobenzidine for 5 minutes. The stained tissue was observed under a microscope.
10
Co-Immunoprecipitation of SYTL1 and FSCN1
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Co-IP assays of SYTL1 and FSCN1 were performed as described in our previous study[31 ] with the Pierce Crosslink Magnetic IP/Co-IP Kit (88805, Thermo Scientific, Shanghai, China). Briefly, 5 μg of an anti-Flag (14793), anti-IgG (3900, CST) or anti-FSCN1 (1:1000, ab126772, Abcam) antibody was bound to Protein A/G Magnetic Beads for 1 h at room temperature. The antibody-crosslinked beads were then incubated overnight at 4 °C with 400 μg of cell lysate that contained Flag-SYTL2 and FSCN1. The proteins interacting with the antibodies were eluted from the magnetic beads. The samples were then used for SDS‒PAGE immunoblot analysis, and the SDS‒PAGE gel was silver-stained by using the Pierce Silver Stain Kit (24612, Thermo Scientific) or subjected to mass spectrometry (MS) analysis with an Easy nanoLC 1200—Orbitrap Fusion (Thermo Fisher, USA).
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