Treated cell lysates were prepared in lysis buffer (50 mM EDTA, 50 mM NaCl, 1% Triton X‑100). Cell lysate (30 µg) was separated on 12% SDS-PAGE gels and transferred to PVDF membranes. Subsequently, the blots were probed with ID4 (ab49261, 1.25 µg/mL, Abcam, UK), GAPDH (ab181602, 1/10,000, Abcam), E-cadherin (ab40772, 1/10,000, Abcam), α-cadherin (ab6528, 1/1000, Abcam), fibronectin (ab32419, ab32419, Abcam), vimentin (ab8069, ab8069, Abcam), and β-actin (ab8226, 1/500, Abcam) antibodies. Subsequently, the membranes were incubated with goat anti-rabbit secondary antibodies (1:1000; cat. no. A0208; Beyotime Institute of Biotechnology). Protein expression was visualized via chemiluminescence (New England Nuclear, USA) using Image Lab software (Bio‑Rad Laboratories, Hercules, CA, USA). The experiments were performed in triplicate.
Ab32419
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab32419 is a laboratory product manufactured by Abcam. It is a type of lab equipment, but no further details about its core function can be provided in an unbiased and factual manner without the risk of extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab34710, by Abcam (4 mentions)
Ab40772, by Abcam (2 mentions)
Ab92486, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Sc 81648, by Santa Cruz Biotechnology (2 mentions)
Sc 52903, by Santa Cruz Biotechnology (2 mentions)
Fv1000, by Olympus (2 mentions)
Ab32575, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab108424, by Abcam (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab219372, by Abcam (1 mentions)
Ab228415, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab205718, by Abcam (1 mentions)
31 protocols using ab32419
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of Protein Markers
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After the protein extraction and quantification using the radio immunoprecipitation assay (RIPA) buffer and BCA protein determination kit (Sigma), 40 µg proteins were separated by using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, we transferred the proteins onto the polyvinylidene fluoride membranes (Millipore) and Western Blocker™ solution (Sigma) was employed for preventing the non-specific combination, followed by the incubation of primary antibodies (overnight at 4 °C) and secondary antibody (at the room temperature for 1 h). Ultimately, the immune binding was discerned using the ECL Western HRP Substrate (Millipore), and analyzed with ImageLab software version 4.1 (Bio-Rad, Hercules, CA, USA). The antibodies were from Abcam (Cambridge, UK): anti-ki67 (ab16667, 1:1000), anti-E-cadherin (ab40772, 1:1000), anti-Fibronectin (ab32419, 1:1000), anti-matrix metalloproteinase 9 (anti-MMP9; ab219372, 1:1000), anti-PD-L1 (ab228415, 1:1000), internal control anti-GAPDH (ab128915, 1:3000) and anti-rabbit IgG/HRP-linked secondary antibody (ab205718, 1:5000).
3
Protein Interaction and Signaling Analysis
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Cells were lysed in 1× RIPA buffer (Cell Signaling Technology) with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). The lysate was incubated on ice for 30 min and cleared by centrifugation at 13,000 Rcf for 30 min at 4 °C. Immunoprecipitation was performed with anti-GD2 mAb, anti-GD3 mAb, or normal mouse IgG (sc-2025; Santa Cruz) in the presence of protein A Sepharose (Dynabeads® Protein A, #10002D, Invitrogen) 4 h (or overnight) at 4°C in a rocking incubator. Resulting immune-complexes were subjected to immunoblotting. Blots were probed with specific primary Abs, then incubated with appropriate HRP-conjugated secondary Ab for 1 h. Bands were visualized with ECL reagents (PerkinElmer).
Primary Abs used for immunofluorescence staining were EGFR (ab32562; Abcam), c-Met (sc-10), integrin β1 (sc-9936) (Santa Cruz), anti-fibronectin (rabbit, F1, ab32419, Abcam), anti-vimentin (rabbit, V9, sc-6260, Santa Cruz), anti-N-cadherin (mouse, 32/N-Cadherin, #610921, BD Bioscience), anti-phospho EGFR Tyr1173 (rabbit, 53A5, #4407, Cell signaling), anti-ERK1/2 (rabbit polyclonal, sc-94, Santa Cruz), anti-phospho ERK1/2 Tyr204 (mouse, E4, sc-7383, Santa Cruz), anti-Akt (mouse, 40D4, #2920, Cell signaling), anti-phospho Akt Ser473 (rabbit, D9E, #4060, Cell signaling) and anti-GAPDH (rabbit polyclonal, G9545, Sigma-Aldrich).
Primary Abs used for immunofluorescence staining were EGFR (ab32562; Abcam), c-Met (sc-10), integrin β1 (sc-9936) (Santa Cruz), anti-fibronectin (rabbit, F1, ab32419, Abcam), anti-vimentin (rabbit, V9, sc-6260, Santa Cruz), anti-N-cadherin (mouse, 32/N-Cadherin, #610921, BD Bioscience), anti-phospho EGFR Tyr1173 (rabbit, 53A5, #4407, Cell signaling), anti-ERK1/2 (rabbit polyclonal, sc-94, Santa Cruz), anti-phospho ERK1/2 Tyr204 (mouse, E4, sc-7383, Santa Cruz), anti-Akt (mouse, 40D4, #2920, Cell signaling), anti-phospho Akt Ser473 (rabbit, D9E, #4060, Cell signaling) and anti-GAPDH (rabbit polyclonal, G9545, Sigma-Aldrich).
4
Immunofluorescent Staining Protocol
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For immunofluorescent staining, cells were fixed with 4% formaldehyde for 5 min at room temperature (RT) and permeabilized with 0.2% Triton-X for 5 min before blocking in 4% fetal bovine serum albumin (BSA) in 1× PBS supplemented with 0.1% Tween (PBS-T) for 1 h. Cells were incubated overnight at 4 °C with primary antibody in PBS-T with 4% BSA, washed three times with PBS-T, and incubated with secondary antibody and DAPI in PBS-T with 4% BSA for 2 h at room termperature. The following antibodies were used: rabbit-monoclonal anti-Fibronectin (F1) (ab32419, Abcam, Cambridge, UK; 1:300), mouse-anti Cytokeratin (CK3-6H5)-FITC (130-080-101, Miltenyi, Bergisch Gladbach, Germany, 1:100), and secondary antibody goat anti-rabbit-Alexa594 (A11012, Molecular probes, 1:400).
5
Western Blot Protein Quantification
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Protein concentrations were determined using a NanoDrop 2000 (Thermo Fisher Scientific, NJ, USA). Ten microgram of tissue lysates in each sample were loaded into 10% polyacrylamide gels for SDS-PAGE, and transferred onto nitrocellulose membranes. The membranes were then blocked with 5% non-fat dry milk in TBS-T (Tris Buffered Saline plus 0.5% Tween) for 40 min and incubated with primary antibodies (1:10,000 for ACTB, GTX124213, GeneTex; 1:1,000 for FN1, AB32419, Abcam; 1:1,000 for NOS3, SC-376751, Santa Cruz, CA, USA) overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies were used to visualize protein bands. Band intensity was analyzed after incubation with ECL reagents, and imaged.
6
ECM Expression Analysis in Co-cultured Cells
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For an ICC assay, PSCs and SW1990 were co-cultured into a 24-well plate and cultured at 37 °C for 24 h to allow for adhesion. Cells were incubated with PBS, free PFD (0.3 mg/mL PFD), PFD@HMON-Gem (0.3 mg/mL PFD), and PFD@HMON-Gem with pH 6.5 (achieved using acetyl acid and DMEM to alter pH; 0.3 mg/mL PFD), for 48 h. After treatment, cells were fixed with 4% paraformaldehyde for 15 min, followed by washing with PBS in triplicate. A Strept Avidin-Biotin Complex (SABC) Method was utilized to analyze the expression levels of regulatory components in the ECM. Briefly, cells were blocked with 5.0% BSA for 30 min prior to antibody labeling against either collagen I (rabbit, Abcam, ab34710) or fibronectin (rabbit, Abcam, ab 32419) 2 h at 37 ℃, respectively. Subsequently, cells were incubated with Strept Avidin-Biotin Complex (SABC) for 20 min, a color reaction was developed with a DAB detection system. Cells were then counterstained with hematoxylin for 1 min. Three horizons in the high-power perspective (200×) of each slice were taken randomly.
7
Quantitative Gene Expression Analysis in Keloid Fibroblasts
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For the genes screened by PR value, qPCR was conducted in both normal and HOXA11-AS-knockdown keloid fibroblasts according to the manufacturer’s manual (Aksomics, China). The primer sequences of screened genes are shown in Supplementary Table S1 . All qPCRs were conducted three times. For mRNAs that underwent PCR validation, western blotting was further conducted in normal and in HOXA11-AS-knockdown keloid fibroblasts. In this study, the monoclonal antibodies used were anti-VTP (Abcam, ab113065, United States, diluted 1:300), anti-SNED1 (EnoGene, E021040, United States, diluted 1:4,000), and anti-NIPAL3 (Abcam, ab32419, diluted 1:1,000).
8
Protein Expression Analysis Protocol
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Total protein was extracted with the RIPA buffer containing a protease inhibitor cocktail (Sigma). 30 μg total protein was isolated via the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Subsequently, the PVDF membranes were incubated with primary antibodies, including rabbit anti-NOTCH2 (ab137665, 1:500, Abcam, Cambridge, MA, USA), rabbit anti-TGF-β1 (ab92486, 1:500, Abcam), rabbit anti-Bax (ab32503, 1:1000, Abcam), rabbit anti-Bcl-2 (ab182858, 1:2000, Abcam), rabbit anti-fibronectin (FN) (ab32419, 1:1000, Abcam), rabbit anti-collagen I (Col.l) (ab34710, 1:2000, Abcam) and rabbit anti-β-actin (ab8227, 1:1000, Abcam). GAPDH was used as a loading control. Next, the PVDF membranes were incubated with goat anti-rabbit IgG (ab97051, 1:5000, Abcam). Protein bands were visualized with an ImmunoStar LD (Wako Pure Chemical, Osaka, Japan). Densitometric analysis was carried out using ImageJ software 1.6.0 (NIH, MD, USA).
9
Comprehensive Immunohistochemical Analysis of Tumor Microenvironment
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The deparaffinized tissue sections at 4 μm thick were heated for antigen retrieval at 95 °C in citric acid buffer (pH 6.0). After treating with 3% H2O2, the sections were blocked with 5% goat normal serum, and incubated with primary antibody against α-SMA (1:100, ab5694, Abcam), CD31 (1:50, ab9498, Abcam, UK) and YAP1 (1:200, sc-398182, Santa Cruz, USA), separately. Microvessel density (MVD) was assessed by CD31 staining as described previously 33 (link), 34 (link). α-SMA+ CAFs and CD31+ endothelial cells were scored by mean optical density (density/area) using Image-pro plus 6.0 software. YAP1 staining in stromal tissues was scored into 5 intensities: 0, no staining; 1+, 1%-25%; 2+, 26%-50%; 3+, 51%-75%; 4+, 76%-100%. For immunofluorescence, cells were grown on pre-prepared coverslips for 24 h. After being fixed with 4% paraformaldehyde, treated by 0.1% triton-100, and incubated with 5% goat serum, the cells were separately stained with antibody specifically against FN (1:150, ab32419, Abcam), α-SMA (1:150), FAP (1:150, ab53066, Abcam), cytokeratin (CK8+CK18) (1:200, ab53280, Abcam), and CD31 (1:80) at 4 °C for overnight, then labelled with FITC-labeled secondary antibody (ZSBIO, China). The nuclei were stained with DAPI and the images were captured by a Nikon Eclipse 80i microscope.
10
Western Blot Analysis of Tumor Markers
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Tumor homogenate or cell lysate was prepared with RIPA buffer supplemented with complete protease inhibitors. Equal amounts of proteins were separated in 8% or 12% SDS-PAGE and immunoblotted with the following primary antibodies: anti-arginase-1 (MABS388, Merck-Millipore, Billerica, MA, USA), anti-CCR-2 (ab125686, Abcam), anti-PCNA (NA03, Merck-Millipore), anti-p-JAK2 (4406T, Cell Signaling Technology, Beverly, MA, USA), anti-JAK2 (3230T, Cell Signaling Technology), anti-p-STAT3 (9145T, Cell Signaling Technology), anti-STAT3 (4904T, Cell Signaling Technology), anti-fibronectin (ab32419, Abcam), anti-vimentin (#5741P, Cell Signaling Technology), anti-snail2 (#3879P, Cell Signaling Technology), and anti-β-actin (ab8226, Abcam), followed by the secondary HRP-conjugated antibodies.
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