Protein extracts from cells or immunoprecipitation samples were prepared using detergent-containing lysis buffer. Total protein (50 µg) was subjected to SDS-PAGE and transferred to PVDF membrane (Millipore). Antibodies against STAT3 (ab119352), p-STAT3 (p-Y705, ab76315), SRC (ab109381), BCL-XL (ab32370), active CASPASE-3 (ab32351), MMP2 (ab86607) or MMP9 (ab58803) were from Abcam. Antibodies against cleaved CASPASE-7 (p20, D198; orb159339) or cleaved CASPASE-9 (p35, D315; orb159343) were from Biorbyt. Antibodies against PIWIL2 (sc-67502) or p-SRC (p-Tyr416, D4964) were from Santa Cruz or Cell Signaling Technology, respectively. Antibody against FLAG tag (F1804) or 6×HIS tag (SAB2702218) was from Sigma, while antibody against β-ACTIN (60008-1-Ig) was from Proteintech. Membranes were incubated overnight at 4 °C with primary antibody and visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore).
Ab58803
Manufactured by Abcam
Sourced in United States, China, United Kingdom
Ab58803 is a recombinant monoclonal antibody. It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab92536, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab86607, by Abcam (2 mentions)
Ab32072, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab2462, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ab109381, by Abcam (1 mentions)
Ab32351, by Abcam (1 mentions)
Ab32370, by Abcam (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ab119352, by Abcam (1 mentions)
Ab68153, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab8978, by Abcam (1 mentions)
Ab22656, by Abcam (1 mentions)
Ab6328, by Abcam (1 mentions)
880 confocal microscope, by Zeiss (1 mentions)
Mowiol 4 88 reagent, by Merck Group (1 mentions)
18 protocols using ab58803
1
Western Blot Analysis of Signaling Proteins
2
Protein Expression Analysis in Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells cultured in the 6-well plate were added to ristocetin-induced platelet agglutination (RIPA) for lysis. The supernatant was centrifuged after the lysis was completed. The BCA kit was used to detect the protein concentration of each group of cells. The target protein sample was heat-denatured before being put into a 12 percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel for electrophoresis separation and transfer to PVDF membranes. After the transfer was completed, the membrane was immersed in 5% skimmed milk powder and sealed for 2 hours. The PVDF membrane was incubated with primary antibodies including MMP-2(ab92536, Abcam, 1:1000 dilution), MMP-9(ab58803, Abcam, 1:1000dilution), STAT3(ab68153, Abcam, 1:800dilution), cyclin D1(ab16663, Abcam, 1:1000dilution), c-Myc (ab32072, Abcam, 1:1000dilution), β-actin(66009-1-Ig, protein each, 1:5000)which was used as a control at 4° C overnight. Shaked and washed 3 times in TBS solution and incubated with secondary antibodies at 24° C for 1h. Immune complexes were detected by the Western bands and were analyzed by ImageJ software.
3
Protein Expression Analysis in NP69 cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NP69 cells were collected at 72 h after lentiviral infection and lysed with lysate. The lysate was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane and blocked with 5% skim milk at room temperature for 2 h. The membrane was incubated, using the following mouse anti human primary antibodies: Anti-E-cadherin, anti-β-catenin, anti-fibronectin, anti-vimentin and anti-MMP9 (dilution, 1:1,500; cat. nos. ab1416; ab22656; ab6328; ab8978; ab58803 respectively, purchased from Abcam plc., Burlingame, CA, USA) at room temperature for 1 h. The membrane was then incubated with HRP-conjugated rabbit anti-mouse secondary polyclonal antibody (dilution, 1:2,500; cat. no. ab6728; Abcam plc.) for 1 h. The HRP enzyme substrate was added and incubated for 5 min to develop color, and the immunoblotting result was recorded using a fully automated western blot WEs analysis system (ProteinSimple, San Jose, CA, USA). The density of each band was determined by ImageJ software, and β-actin was used as the internal reference. CNE1 and NP69 cells infected with blank lentivirus were used as controls. The relative expression level was calculated as the ratio of the assessed protein and β-actin and expressed as the mean ± standard deviation (n=3).
4
Immunostaining Protocol for MMP9 and Cullin-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissues and hepatocytes were treated with 4% PFA/PBS for 30 min at room temperature. Then, samples were treated with 0.25% Triton-X100/PBS for 30 min, followed by the incubation with 4% BSA/PBS for 45 min 22 (link). Primary antibodies against MMP9 (1:1000, #ab58803, Abcam) and Cullin-1 (1:1000, #ab202555, Abcam) and suitable fluorescently-labeled secondary antibodies were sequentially incubated for 2 h, and nuclear staining performed with 5 μg/ml Hoechst 33342 before mounting in Mowiol 4-88 reagent (81381, Sigma). Fluorescence images was captured with a Zeiss 880 confocal microscope using a 100X oil objective, and acquired with same laser output and gain 45 (link).
5
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted using the RIPA buffer (Beyotime). Western blotting was done as described by Chen et al. [33 (link)] with specific primary antibodies against SIX1 (ab243247, Abcam, USA), matrix metalloproteinase 9 (MMP9) (ab58803, Abcam), and Cleaved-caspase-3 (Cleaved-caspase-3, Abcam), and β-Actin (ab8226, Abcam). Development of the blots was done with the SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher).
6
Histological Analysis of Xenograft Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The xenograft tumor tissues and lung tissues of mice were fixed with 10% paraformaldehyde, embedded in paraffin, and then cut into sections of 4 μm thickness. The sections were dewaxed using xylene and rehydrated using successive gradient concentrations of ethanol. For H&E staining, the lung tissue sections were first stained with hematoxylin and then with eosin. Then the sections were dehydrated and sealed. For IHC analysis, xenograft tumor tissue sections were treated with 3% H2O2 for 10 min at room temperature to inactivate the endogenous enzyme. The sections were immersed in a 0.01 M citrate buffer (pH = 6.0) and heated to boiling in a microwave oven. After cooling, samples were blocked with 5% BSA blocking solution at room temperature for 20 min. Then, the sections were incubated with Ki-67 (1:100, ab156956, Abcam, Shanghai, China) and matrix metalloproteinase-9 (MMP-9) (1:100, ab58803, Abcam, Shanghai, China) antibodies overnight at 4 °C. Subsequently, the sections were incubated with goat anti-rabbit IgG antibody (1:200, ab6721, Abcam, Shanghai, China) for 20 min at 25 °C. Diaminobenzidine (DAB) staining and hematoxylin counterstaining were performed. Sections were dehydrated and sealed in neutral resin. Images were captured using a light microscope (Olympus BX40F, Olympus, Tokyo, Japan).
7
Comprehensive Immunohistochemical Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, the sections had been repaired with the antigen repair solution, observed by using 0.5% Triton X-100 permeabilized, and blocked with 5% (w/v) bovine serum albumin (BSA). Then the sections were incubated with primary antibodies at 4°C overnight. Primary antibodies used were as follows: TGF-β1 (ab215715, Abcam, 1:200), MMP9 (ab58803, Abcam, 1:200), Platelet endothelial cell adhesion molecule-1 (CD31) (M1511-8, Huabio, 1:200), Cleaved caspase-3 (ab179517, Abcam, 1:200), NKG2-D (sc-515599, Santa Cruz, 1:200), and nucleotide-binding oligomerization domain containing 2 (NOD2) (sc-56168, Santa, 1:200). The slides were incubated with secondary antibodies for 60 min. Finally, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (c0060, Solarbio), and the slides were visualized by fluorescence microscopy.
8
Immunohistochemical Analysis of Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical studies of the lung tissue were conducted using the following antibodies: Monoclonal rabbit anti-mouse vascular endothelial growth factor (VEGF; ab1316), cluster of differentiation (CD34; ab81289), proliferating cell nuclear antigen (PCNA; ab92552), and MMP9 (ab58803) (dilution 1:400), which were all purchased from Abcam (Cambridge, UK); biotinylated goat anti-rabbit IgG was used as the secondary antibody. An S-P kit (Gene Tech Biotechnology Co., Ltd., Shanghai, China) was used for the immunohistochemistry experiments. Briefly, the paraffin sections were deparaffinized in xylene and rehydrated in an alcohol gradient. Antigen retrieval was performed by microwave treatment in 0.01 M sodium citrate buffer (pH=6.0) for 15 min, after which the slides were cooled to room temperature for 1 h, and subsequently washed with PBS. Endogenous peroxidase activity was inactivated with 0.3% hydrogen peroxide for 30 min and non-specific protein interactions were blocked with an avidin/biotin blocking solution containing 10% normal goat serum. The sections were subsequently incubated overnight with primary antibodies against VEGF, CD34, PCNA, and MMP9. The immunoreactions were detected after staining with 3,3′-diaminobenzidine.
9
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA buffer (pH = 8.0) including 150 mM NaCl, 0.1% SDS, 50 mM Tris-HCl and 1% NP-40 was added to the proteinase inhibitor (Roche Diagnostics, Co., Ltd., Rotkreuz, Switzerland) and used to obtain whole cell lysates. The proteins were examined using a BCA kit (Thermo Fisher) and separated by SDS-PAGE. The proteins were transferred onto PVDF membranes (0.45 µm, Millipore Corp, Billerica, MA, USA), sealed with 5% BSA for 60 min at 25°C, and hybridized at 4°C with primary antibodies to MMP2 (1:1000, MA5-14,186, Invitrogen Inc., Carlsbad, CA, USA), MMP9 (1:2000, ab58803, Abcam), Bax (1:1200, sc-7480, Santa Cruz Biotechnology), PI3K (1:1,800, sc-8010, Santa Cruz Biotechnology), p-AKT (1:1,500, ab81283, Abcam), AKT (1:1,300, sc-5298, Santa Cruz Biotechnology), and GAPDH (1:3,000, G8795, Sigma-Aldrich, St Louis, MO, USA) for one night. The blots were incubated with secondary antibodies for 60 min at 25°C. Finally, the protein bands were detected using an ECL kit (Thermo Fisher Scientific). Densitometric analysis was performed using IPP 6.0 (Image-Pro Plus 6.0).
10
Immunohistochemical Analysis of MMPs and Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SP method was used for immunohistochemistry according to the manufacture’s protocol. After incubation with 1% H2O2 in methanol and 10% goat serum for 30 min, the primary antibodies to MMP2 (ab2462, Abcam, Hong Kong, China), MMP9 (ab58803, Abcam, Hongkong, China), and monoclonal mouse anti-rabbit macrophage (Clone RAM11, Dako, Denmark) were incubated overnight at 4 °C. Sections were incubated with biotinylated anti-mouse second antibody for 20 min, and visualized with diaminobenzidine tetrahydrochloride. Immunofluorescence was used to detect the assembly of smooth muscle cells (SMCs). Sections were incubated overnight with mouse monoclonal anti-alpha-smooth muscle actin (A2547, Sigma-Aldrich (Shanghai) Trading Co., Ltd, China), and subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse IgG. Semiquantitative analyses for SMCs content was calculated as the mean area of 6 fields per rabbit.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!