mPFBs were cultured on plates of different elastic modulus (8 kPa, 0.2 kPa) coated with 100 µg/ml collagen type I (PureCol, Advanced BioMatrix, Carlsbad, CA) in DPBS for 24 h. Cell lysates were harvested with RIPA buffer (Sigma Aldrich) containing protease and phosphatase inhibitor cocktails. Protein concentration was determined by Pierce® BCA Protein assay kit (Pierce; Waltham, MA). 12 µg proteins of each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and blocked with 5% dried milk in TBS-T or 5% bovine serum albumin (BSA) in TBS-T. Primary antibodies for fibronectin (ab1954, 1:2000, Millipore), α-SMA (A5228, 1:2000, Sigma Aldrich), pFAK Y397 (ab81298, 1:1000, Abcam), FAK (ab40794, 1:1000, Abcam), Vinculin (MAB3574, 1:1000, Millipore), β1-integrin (SAB5600100, 1:1000, Sigma-Aldrich), GAPDH (MAB374, 1:1000, Millipore) were used to incubate the membranes for 12 h. Detection was with appropriate peroxidase-conjugated secondary antibodies (1:2500, Jackson ImmunoResearch; West Grove, PA), which were developed with Clarity Western ECL substrate (Bio-Rad; Hercules, CA). Densitometry analysis was performed using Image Lab Software (Bio-Rad).
Mab3574
Manufactured by Merck Group
Sourced in United States
MAB3574 is a monoclonal antibody that can be used as a laboratory reagent. Its core function is to detect and bind to a specific target molecule or antigen. The detailed specifications and intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Purecol, by Advanced BioMatrix (2 mentions)
A5228, by Merck Group (2 mentions)
Ab129068, by Abcam (2 mentions)
Image lab 6, by Bio-Rad (2 mentions)
Ab201060, by Merck Group (2 mentions)
Nupage lds sample loading buffer, by Thermo Fisher Scientific (2 mentions)
Hyglo chemiluminescent hrp detection reagent, by Thomas Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Specific bottom glass dishes, by MatTek (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Alexa fluor 594 conjugated phalloidin, by Thermo Fisher Scientific (2 mentions)
Tween 20, by Merck Group (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ab40794, by Abcam (1 mentions)
Ab81298, by Abcam (1 mentions)
Mab374, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
15 protocols using mab3574
1
Mechanosensitive Protein Expression in mPFBs
2
Antibody Detection Protocols for p53 Signaling
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Antibodies used in this paper are listed here: anti‐P53 antibody (SC‐126; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anticaspase‐3 (SC‐7148; Santa Cruz Biotechnology, Inc.), antivinculin antibody (MAB3574; Millipore, Bedford, MA, USA), anti‐FEN1 (70185; GeneTex, Inc., Irvine, CA, USA), antitubulin (AM103a; Bio‐world, Dublin, OH, USA), anti‐GAPDH (AP0063; Abgent, Suzhou, China), anti‐γ‐H2AX (ab2893; Abcam, Cambridge, MA, USA), anticleaved caspase‐3 antibody: (Asp175) antibody #9661 (Cell Signaling Technology, Danvers, MA, USA), antiphospho‐P53: phospho‐p53 (Ser15) antibody #9284 (Cell Signaling Technology), anti‐Myc‐tag (AP0031M; Abgent), P53BP1 (SC‐22760; Santa Cruz), Alexa Fluor ®488 goat anti‐rabbit A‐11008 Life Technologies, Alexa Fluor ®594 donkey anti‐rabbit R37119 Life Technologies.
3
Immunoblotting of Brain Protein Markers
4
Immunofluorescence Staining of Cell Junctions
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Cells were fixed in 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton X-100 in phosphate-buffered saline (PBS), and blocked in 5% bovine serum albumin (BSA). Samples were subsequently incubated overnight in primary antibody in 1% BSA/0.3% Triton X-100/PBS, followed by washing in PBS and incubation in secondary antibody in 1% BSA/0.3% Triton X-100/PBS. Finally, samples were mounted in Elvanol. The following antibodies were used: VE-cadherin (Abcam AB33168; 1:400), β-catenin (Santa Cruz Biotechnology; sc-7199; 1:500) Phospho-Myosin Light Chain 2 (Thr18/Ser19; Cell Signaling; 3674; 1:200), vinculin (Millipore MAB 3574, 1:500), α18 (Yonemura et al., 2010 (link)), 1:8000), HA-Tag (Cell Signaling 2367; 1:1000) streptavidin (ThermoFisher Scientific MA1-20010; 1:1000), and Alexa Fluor 488, 568, and 647 conjugated secondary antibodies (Invitrogen; 1:400). Actin was labeled with Alexa Fluor 488, 568, or 647-conjugated phalloidin (Invitrogen; 1:600).
All fluorescence images were collected by laser scanning confocal microscopy (SP8X; Leica) with Leica Application Suite software (LAS X version 2.0.0.14332), using 40×, 63×, or 100× immersion objectives.
All fluorescence images were collected by laser scanning confocal microscopy (SP8X; Leica) with Leica Application Suite software (LAS X version 2.0.0.14332), using 40×, 63×, or 100× immersion objectives.
5
Characterization of Myofibroblast Phenotype
6
Quantifying Cell Adhesion via Focal Contact Analysis
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Cell adhesion was determined through the analysis of focal contacts by actin filaments and vinculin detection. In these studies, 100,000 cells were seeded onto the different samples and, after 72 h, alloys were washed twice in PBS and cells fixed in 4% paraformaldehyde in PBS for 15 min at RT. After washing twice in PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 15 min and blocked for 25 min with 1% bovine serum albumin (BSA) (Sigma-Aldrich), 0.5% Tween 20 (Sigma-Aldrich) in PBS at RT. Samples were then incubated with a mouse anti-vinculin primary antibody (Millipore, MAB3574) at 2 µg/mL overnight at 4 °C and washed with 1% BSA-0.5% Tween 20 in PBS. Then, samples were incubated with a mixture of Alexa fluor 594-conjugated phalloidin (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), Alexa fluor 488 chicken anti-mouse IgG (Invitrogen), and Hoechst 33,258 (Sigma-Aldrich) for 60 min in the dark at RT. Finally, cells were washed in PBS, air-dried, and mounted on specific bottom glass dishes (MatTek, Ashland, MA, USA) using ProLong Antifade mounting solution (Life Technologies, Carlsbad, CA, USA). Immunofluorescence evaluation was performed in a confocal laser scanning microscope (CLSM) (Olympus, Shinjuku, Tokyo, Japan).
7
Immunoblotting of Brain Protein Markers
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The cortex from E4FAD mouse brains was homogenized in Eppendorf tubes (kept on ice), using 300 ul of RIPA buffer (Thermo Fischer Scientific) with freshly added protease and phosphatase inhibitor cocktails. The samples were placed on an orbital shaker at 4°C overnight and spun the next day at 12000 rpm for 20 min at 4°C. The supernatant from each sample was collected and the protein concentration was assayed using BSA as working standard. Equal amounts of protein (40 μg) were heat-denaturized in NuPAGE LDS sample- loading buffer (Invitrogen) for 5 min at 95°C, resolved by SDS-PAGE and transferred to PVDF membranes (Sigma-Aldrich, MO, USA). The membranes were blocked with Tris-buffered saline (TBS) containing 0.05% Tween and 5% non-fat dry milk (NOX2, Aβ42) and Tris-buffered saline (TBS) containing 0.05% Tween and 2% BSA (Vinculin) and then incubated overnight with antibodies directed against NOX2 (Rabbit monoclonal, 1:5000, Abcam, ab129068), Aβ42 (Rabbit monoclonal, 1:500, Abcam, ab201060) or Vinculin (Mouse monoclonal, 1:1000, Millipore, MAB3574). Peroxidase conjugated IgG was used as secondary antibody. Membrane-bound immune complexes were detected by HyGLO™ Chemiluminescent HRP Detection Reagent (Denville Scientific). Protein loading was normalized according to Vinculin expression. Quantification was performed by densitometric analysis using Imagelab 6.0 software (Bio-Rad).
8
Comprehensive Immunostaining Protocol for Huntington's Disease
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The following primary antibodies were used: anti‐HTT (mEM48) antibody (1:50, MAB5374, Millipore); anti‐polyglutamine antibody (1C2) (1:2000, MAB1574, Millipore); anti‐Huntingtin antibody(EPR5526)(1:5000, ab271195, Abcam); anti‐vinculin antibody (1:1000; MAB3574, Millipore); anti‐NeuN antibody (1:1000, ab177487, Abcam); anti‐GFAP antibody (1:5000, ab7260, Abcam); and anti‐Iba1 antibody (1:1000, 019–19741, Wako); anti‐P62 antibody (1:1000, ab56416, Abcam); anti‐LAMP1 antibody (1:1000, ab24170, Abcam); anti‐LAMP2 antibody (1:1000; 49067S, Cell Signaling Technology); anti‐LC3A/B antibody (1:1000; 4108S, Cell Signaling Technology); Anti‐GFP antibody (1:1000, A11122, Invitrogen); anti‐HA tag antibody (1:100; 3724S, Cell Signaling Technology); anti‐Beclin 1 antibody (1:1000; 3738S, Cell Signaling Technology)
9
Western Blot Protein Detection
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Resolved proteins were transferred to a membrane (Sequi-Blot PVDF, 0.2 µm; Bio-Rad) in transfer buffer, pH 8.3 (25 mM Tris, 192 mM glycine, 0.05% SDS, 20% methanol), for 12 h at 30 V and 15 ºC (Criterion™ Blotter; Bio-Rad). The membranes were blocked twice in 5% non-fat milk dissolved in TBST (pH 7.5, 150 mM NaCl, 20 mM Tris-HCl, 0.1% Tween-20) for 30 min each time, and then rinsed three times in TBST for 5 min each time. Each blocked membrane was incubated for 1.5 h at 37 °C with monoclonal anti-desmin (DE-U-10; Sigma Aldrich) diluted 1:200 in TBST containing 0.5% non-fat milk. The membranes were washed for 10 min with TBST containing 0.5% non-fat milk, rinsed twice with TBST for 5 min each time, and incubated with IgG-HRP sc-2005 anti-goat (1:10,000; Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal antivinculin (1:1,000, MAB3574; Millipore, Billerica, MA, USA), or monoclonal anti-myosin (1:200, MY-32; Sigma Aldrich) for 1 h at 20 °C. Following incubation with the appropriate secondary antibody, the membranes were rinsed with TBST for 10 min and then with TBS (pH 7.5, 150 mM NaCl, 20 mM Tris-HCl) for 5 min. The protein spots were visualized using an Immobilon Western Chemiluminescent HRP kit (Millipore) with incubation for 10 min, and exposed to film (Logic 1500 Kodak; Imaging System).
10
Visualizing Focal and Fibrillar Adhesions
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Cells were fixed with 4% paraformaldehyde for 5 min, rinsed in
1X phosphate buffered saline, incubated within 0.1% Triton X-100 solution
for 5 min, rinsed, and blocked by a 1% bovine serum albumin solution
(Millipore Sigma) for 30 min. Focal adhesions were visualized using
antibodies specific for vinculin (MAB3574; Millipore Sigma) and integrin
αvβ3 (MAB1976Z; Millpore Sigma). Fibrillar adhesions were
identified using specific antibodies to tensin-1 (NBP1–84129;
Novus biologicals, Toronto, ON), integrin β1 (MAB17781; R&D
Systems, Minneapolis, MN), and integrin α5 (ab150361; Abcam,
Waltham, MA). Extracellular matrix deposition was identified using
antibodies to fibronectin (ab1954; Abcam, Waltham, MA). F-actin was
labeled with rhodamine conjugated phalloidin (R415; Life Technologies,
Grand Island, New York), and nuclei were counterstained with Hoechst
(H3560; Invitrogen, Burlington, ON). Images were taken on a Carl Zeiss
Axio Imager M2m microscope (Zeiss Microscopes, North York, ON) under
reverse osmosis water immersion and processed using Zen Pro software.
1X phosphate buffered saline, incubated within 0.1% Triton X-100 solution
for 5 min, rinsed, and blocked by a 1% bovine serum albumin solution
(Millipore Sigma) for 30 min. Focal adhesions were visualized using
antibodies specific for vinculin (MAB3574; Millipore Sigma) and integrin
αvβ3 (MAB1976Z; Millpore Sigma). Fibrillar adhesions were
identified using specific antibodies to tensin-1 (NBP1–84129;
Novus biologicals, Toronto, ON), integrin β1 (MAB17781; R&D
Systems, Minneapolis, MN), and integrin α5 (ab150361; Abcam,
Waltham, MA). Extracellular matrix deposition was identified using
antibodies to fibronectin (ab1954; Abcam, Waltham, MA). F-actin was
labeled with rhodamine conjugated phalloidin (R415; Life Technologies,
Grand Island, New York), and nuclei were counterstained with Hoechst
(H3560; Invitrogen, Burlington, ON). Images were taken on a Carl Zeiss
Axio Imager M2m microscope (Zeiss Microscopes, North York, ON) under
reverse osmosis water immersion and processed using Zen Pro software.
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