The lysates from A549 and HCC827 cells were collected by RIPA buffer. After that, a total of 20 μg of protein was transferred onto PVDF membrane, followed by incubation with anti-MMP2 (1/5000 dilution, #ab2462, Abcam), anti-MMP9 (1/2000 dilution, #ab76003, Abcam), anti-fibronectin 1 (1/8000 dilution, #ab23750, Abcam), and anti-GAPDH (1/10000 dilution, #ab181602, Abcam) at 4 °C overnight. The next day, the membrane was washed strictly and probed with secondary antibody and visualized. IHC was performed in tissue microarrays (TMA) containing 58 LUAD tissue samples using anti-fibronectin 1 (1/500 dilution, #ab23750, Abcam).
Ab23750
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab23750 is a high-quality antibody product offered by Abcam. This antibody is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab5694, by Abcam (7 mentions)
Ab76003, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Quantity one analyzer, by Bio-Rad (2 mentions)
Carl imager m2m microscope, by Zeiss (2 mentions)
Ab14041, by Abcam (2 mentions)
Ab14683, by Abcam (2 mentions)
Sc 365684, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
A11008, by Thermo Fisher Scientific (2 mentions)
A11034, by Thermo Fisher Scientific (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Biolux gaussia luciferase assay kit, by New England Biolabs (2 mentions)
Gaussia luciferase, by GeneCopoeia (2 mentions)
Accel sirnas, by Horizon Discovery (2 mentions)
Hpa002926, by Merck Group (2 mentions)
Ab11575, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab34710, by Abcam (2 mentions)
Ab16667, by Abcam (2 mentions)
36 protocols using ab23750
1
Protein Expression Analysis in Lung Cancer
2
Renal Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Renal protein was extracted from the medulla and cortex of rat kidneys. The protein concentration was measured by the Bradford method, and the supernatant was denatured at 95 °C for 5 min in Laemmli sample buffer. Samples were subjected to SDS-PAGE gels. After electrophoresis, proteins were electro-transferred to a polyvinylidene difluoride membrane (Merck), which was incubated in the blocking buffer (5% non-fat milk, 20 mM Tris-HCl, 150mMNaCl, PH = 8.0, 0.01%Tween 20) for 1 h at room temperature and was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam) or anti-Collagen-I (1:1000, sc-293,182, Santa Cruz) or anti-CTGF (1:1000, sc-373,936, Santa Cruz) anti-nNOS (1:1000, 4236 s, CST) or anti-HIF-1α (1:1000, ab2185, Abcam) or anti-VEGF (1:1000,A0280, Abclonal) overnight at 4 °C. Binding of the primary antibody was detected by an enhanced chemiluminescence method (BeyoECL Star, P0018A, Byotime) using horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG,1:1000, Proteintech). The quantification of protein expression was performed using Quantity One Analyzer (Bio-Rad).
3
Immunofluorescence Staining of Fibronectin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections were deparaffinized and permeabilized with 0.1% Triton X-100 (Caledon; Georgetown, ON) PBS, blocked with 10% horse serum in 0.1% Triton X-100 PBS, and incubated with fibronectin (ab23750; Abcam; 1:100) primary antibody for 8–12 hours at 4 °C. Primary antibodies were detected using Cy5-conjugated anti-rabbit (Molecular Probes; Carlsbad, CA; 1:200). All sections were counterstained with Hoechst 33342 dye (1:5000) for nuclei. Images were taken on Carl Zeiss Imager M2m microscope (Carl Zeiss; Jena) using ZenPro 2012 software.
4
Antibody-based Analysis of Fibrosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used for Western blotting and immunofluorescence were as follows: anti-α-SMA (ab5694; Abcam, Cambridge, England), anti-collagen Type I (600-401-103-0.1; Rockland, Inc., PA, USA), anti-fibrinogen (ab23750, Abcam), anti-Periostin (ab14041, Abcam), anti-Smad2 (#3103S; Cell Signaling, Beverly, MA, USA), anti-phosphorylated Smad2 (#3101S; Cell Signaling), anti-Stat3 (#9139S; Cell Signaling), and anti-phosphorylated Stat3 (#9145S, Cell Signaling). Antibodies for the immunohistochemistry experiments were as follows: anti-E-cadherin mAb (M3612; Dako, Carpinteria, CA, USA), anti-α-SMA (ab5694; Abcam), and anti-Ki67 (M7240; Dako). Transforming growth factor (TGF)-β1 was purchased from R&D Systems (240-B; Minneapolis, MN, USA). For in vitro studies, PFD was provided by Shionogi & Co., Ltd. (Osaka, Japan) and dissolved in DMSO to a concentration of 50 μg/mL. For in vivo studies, Pirespa tablets were purchased from Shionogi & Co. and dissolved in DMSO, using one-fifth of the final volume of the total solvent, after crushing using a micro smash (TOMY SEIKO CO., LTD, Tokyo, Japan). Sterile normal saline at four-fifths of the final total volume was then added to bring PFD to a final concentration of 40 mg/mL.
5
Embryonic and Postnatal Tendon Development
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From WT and Scx-Cre::Mmp14 lox/lox mice, whole hind limbs from E15.5 embryos, lower hind limbs without the skin from P0 pups and dissected Achilles tendons from P10 pups were cryo-preserved in OCT-embedding matrix (Thermo Scientific). Longitudinal sections of 8-µm thickness were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min and then blocked with 2% BSA in PBS for 1 hr. FN was detected using a rabbit polyclonal antibody (ab23750; Abcam; diluted 1:500), and periostin was detected using a goat polyclonal antibody (ab14041; Abcam; diluted 1:500). Cy3-conjugated secondary antibodies (Invitrogen) were used and sections were mounted using Vector Shield containing DAPI (Vector Laboratories). Fluorescent images were taken using a digital camera attached to an Olympus BX51 and captured using MetaVue imaging software (Molecular Devices).
6
Immunofluorescence Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Slide-mounted sections of each mouse model were deparaffinized in xylene and rehydrated using a graded series of ethanol. The sections were treated using citrate buffer for antigen retrieval and blocked using 10% normal goat serum. The localization of each protein was probed using the relevant primary antibody: polyclonal anti-vimentin (B2312; Santa Cruz Biotechnology), anti-fibronectin (ab23750; Abcam, Cambridge, MA, USA), anti-collagen (ab34712; Abcam) and anti-BiP (ab53068; Abcam) antibody (diluted 1:100 in assay buffer) according to the manufacturers' instructions. Simultaneously, sections were incubated with 12G1 antibody (1:50). The secondary antibodies Alexa488-anti-mouse and Alexa594-anti-rabbit (Thermo Fisher, Rockford, IL, USA) were used to label citrullination and target proteins, respectively. Protein expression was quantified based on staining intensity. Immunofluorescence photomicrographs were acquired using a Leitz Diaplan microscope (Leica) coupled to the Leica/Wild MPS48 automated photographic system.
7
Immunohistochemical and Immunofluorescence Analysis of Tumor Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor tissues were fixed with 4% paraformaldehyde and then sectioned into 4 μm of sections. IHC was performed according to protocols of the manufacturor. The sections were incubated with rabbit anti-MMP2, MMP9, p-ATM, GLUT1, PKM2 and TGFβ1 polyclonal antibody (1:200, Bioworld) overnight at 4 °C. Then, the sections were sequentially incubated with polyperoxidase-anti-rabbit IgG (ZSBiO) for 30 min at 37 °C, then stained with diaminobenizidine.
Immunofluorescence staining was done following the standard protocol as described previously [16 ]. The primary antibodies specifically against FN (ab23750, abcam,1:200), α-SMA (ab5694, abcam,1:200), ATM (ab47575, abcam, 1:200), p-ATM (ab19304, abcam, 1:200), γH2AX (5883, CST, 1:200), 53BP1 (ab175933, abcam, 1:200), GLUT1 (ab14683, abcam, 1:200), PKM2 (sc365684, Santa Cruz, 1:150) were used. Normal rabbit IgG was the negative control. IHC and IF images were captured using a Nikon Eclipse 80i microscope (Tokyo, Japan).
Immunofluorescence staining was done following the standard protocol as described previously [16 ]. The primary antibodies specifically against FN (ab23750, abcam,1:200), α-SMA (ab5694, abcam,1:200), ATM (ab47575, abcam, 1:200), p-ATM (ab19304, abcam, 1:200), γH2AX (5883, CST, 1:200), 53BP1 (ab175933, abcam, 1:200), GLUT1 (ab14683, abcam, 1:200), PKM2 (sc365684, Santa Cruz, 1:150) were used. Normal rabbit IgG was the negative control. IHC and IF images were captured using a Nikon Eclipse 80i microscope (Tokyo, Japan).
8
Western Blotting Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting analysis was performed as described previously [11 (link)]. Briefly, total cell proteins were obtained using RIPA lysis buffer (P0013B, Beyotime, China), quantified with the BCA protein assay kit (P0012, Beyotime). 50 μg of total proteins were separately electrophoresed in 8%–12% SDS-PAGE gel, subsequently incubated with appropriate primary antibodies as followings: FN (ab23750, abcam,1:1000), FAP (ab53066, abcam,1:1000), α-SMA (ab5694, abcam,1:1000), ATM (2873, CST, 1:1000), p-ATM (5883, CST, 1:1000), γH2AX (9718, CST, 1:1000), CHK2-T68 (ab32148, abcam, 1:1000), Na+/K+ ATPase (ab58457, abcam, 1:800), Hsp90 (ab13492, abcam, 1:800), AKT (4685, CST, 1:1000), p-AKT (12694 s, CST, 1:1000), GLUT1 (ab14683, abcam, 1:500), p-ST/Q (6966 s, CST, 1:1000), PKM2 (sc365684, Santa Cruz, 1:500), MCT4 (ab74109,1:1000), MCT1 (ab90582,1:1000) TGFβ1 (ab675195, abcam, 1:1000), P38 (bs4635, bioworld, 1:1000), p-P38 (bs3566, bioworld, 1:1000), MMP2 (ab92538, abcam, 1:800), and MMP9 (ab76003, abcam, 1:800), GLUT3 (ab41525,1:800), HK2 (ab104836,1:800), HPI (ab86950,1:1000), LDHA (ab101562,1:1000). The appropriate horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG (ZSGBBIO, China) was used as secondary antibodies. The protein bands were visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Tokyo, Japan).
9
Immunofluorescence Staining of Cell Adhesion and Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed in 4% paraformaldehyde (or 100% ice-cold methanol for γ-tubulin) for 10 minutes and permeabilized in 0.2% triton X-100 for 10 minutes at room temperature. Cells were blocked in 0.2% BSA for 1 hour and stained overnight with primary antibodies: anti-N-cadherin (rabbit-anti-mouse, 1:800, 18203; Abcam), anti-γ-tubulin (rabbit-anti-mouse, 1:800, ab11317; Abcam), anti-fibronectin (rabbit-anti-mouse, 1:1000, ab23750; Abcam), and anti-cleaved-caspase 3 (CC3; rabbit-anti-mouse, 1:1000, 9661; Cell Signalling Technologies). All antibodies used were polyclonal and raised in rabbit against mouse epitopes. Cells were washed thrice with PBS and stained with 4′,6-diamidino-2-phenylindole (10236276001; Roche), phalloidin 488 or 568 (A12379, A12380; Invitrogen), or secondary antibodies: goat-anti-rabbit Alexa488 (A-11008; Invitrogen), goat-anti-rabbit Alexa568 (A-11011; Invitrogen) at 1:1000. Coverslips with cells were washed and mounted on slides in ProLong Gold antifade mounting media (P10144; Invitrogen). Images were obtained using a laser-scanning confocal microscope (Olympus FV3000).
10
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After washed with PBS, cells were lysed with RIPA lysate (Beyotime, Shanghai, China). Protein concentrations were determined using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein was resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membrane (Invitrogen, USA). Afterwards, membranes were incubated with primary antibodies (Anti‐E‐Cadherin, ab1416, 1:50; Anti‐Mucin‐1, ab109185, 1:1000; Anti‐Vimentin, ab8978, 1/100; Anti‐Fibronectin, ab23750, 1 μg/mL; Anti‐c‐Myc, ab39688, 1/500; Anti‐β‐Actin, ab11003, 1/500, Abcam, Cambridge, MA, USA; Anti‐MAPK1, 1:100, Bosterbio, USA). Subsequently, we added IgG‐HRP labelled goat anti‐mouse secondary antibody (ab205719, 1:10000, Abcam, Cambridge, MA, USA). The immunoreactive proteins were visualized using the ECL Detection System (Life technologies, Gaithersburg, MD, USA).
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!