Adherent cells were seeded at 1.5 x 105 cells in two 60‐mm Petri dishes and grown to subconfluence, and total 3 x 105 cells of suspension cell pellet were collected. Whole‐cell protein extracts were prepared by lysing the cells in Laemmli buffer, followed by sonication and boiling for 3 min. Cells were normalised for cell number. After denaturation, proteins were separated by SDS‐PAGE before wet transfer onto PVDF membrane, which were blocked with 5% milk in Tris‐buffered saline (Bio‐Rad Laboratories, Watford, Hertfordshire), containing 0.1% Tween‐20 (TBS‐T, Sigma‐Aldrich). Blocked membranes were probed with IL‐18 antibody (clone 25‐2G, 1 µg mL−1) or E‐cadherin (clone 180224, 2 µg mL−1) in TBS‐T plus 5% milk overnight at 4°C, washed several times with TBS‐T and incubated for 1 h with anti‐rabbit or anti‐mouse peroxidase‐conjugated antibody (1:1000; Dako/Agilent Technologies). Western blots were visualised using an enhanced chemiluminescence kit according to the manufacturer's instructions (GE Healthcare, Buckinghamshire). Signals were then visualised using ImageQuant LAS 4000 (GE Healthcare). The band intensity was quantified by densitometric analyses using the ImageJ software.
Tbs t
Manufactured by Agilent Technologies
Sourced in Denmark
The TBS-T is a lab equipment product from Agilent Technologies. It is a tool used for the transfer and blotting of proteins or nucleic acids from a gel to a membrane for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (4 mentions)
Fluoro gel mounting medium, by Electron Microscopy Sciences (4 mentions)
Tbs t, by Merck Group (3 mentions)
Image reader las 3000, by Fujifilm (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Tbs t, by Santa Cruz Biotechnology (2 mentions)
Oregon green 488, by Thermo Fisher Scientific (2 mentions)
Antibody diluent solution, by Agilent Technologies (2 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Tris buffered saline (tbs), by Bio-Rad (1 mentions)
Las 4000, by Cytiva (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Sodium azide, by Agilent Technologies (1 mentions)
3diaminobenzidine, by Agilent Technologies (1 mentions)
Automation hematoxylin, by Agilent Technologies (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Lumilight reagent, by Roche (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
12 protocols using tbs t
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Protein Phosphorylation Analysis
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Samples were separated onto 10% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane in 10 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS, pH 11, Roth, Karlsruhe, Germany). The membranes were blocked in 5% milk in Tris-buffered saline with 0.1% Tween 20 (Sigma-Aldrich) (TBS-T, 137 mM NaCl, 20 mM Tris base, pH 7.4, 0.1% Tween 20) for 1 h and incubated with primary antibody, monoclonal mouse anti-rat pSer202/pT205 overnight at 4 °C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody in TBS-T (1:2000, Dako, Glostrup, Denmark) for 1 h at room temperature. Immunoreactive proteins were detected by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific, Pittsburgh, PA, USA) and the signals were digitized by Image Reader LAS-3000 (FUJIFILM, Bratislava, Slovakia).
3
Immunohistochemical Detection of Hypoxic Cells
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Antigen retrieval was performed by incubating the sections in citrate buffer (Target Retrieval Solution; Dako) at 90°C for 30 min. Sections were then washed in Tris-buffered saline (154 mM NaCl) with Tween 20 (TBST; Dako) once they had cooled to 80°C. Excessive tissue peroxidase activity was then quenched using 0.03% vol/vol hydrogen peroxide containing sodium azide (Dako) for 10 min. Sections were then incubated in a protein block serum (Protein Block Serum-Free; Dako) for 10 min, to remove nonspecific binding, and washed twice more in TBST. Sections were then treated with an affinity-purified polyclonal anti-pimonidazole antibody raised in the rabbit (1:200 dilution, PAb2627AP; Hydroxyprobe) for 1 h at room temperature before incubation in goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP; polyclonal goat, EnVision; Dako) for 30 min at room temperature. Sections were washed twice with TBST before incubation with 3-diaminobenzidine (Dako) for 10 min and then counterstained with hematoxylin (Automation Hematoxylin; Dako) before coverslips were mounted.
4
Western Blot Analysis of Phosphorylated Tau
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Samples were separated
into 12% SDS-polyacrylamide gels and transferred to a nitrocellulose
membrane in 10 mM N-cyclohexyl-3-aminopropanesulfonic
acid (CAPS, pH 11, Roth, Karlsruhe, Germany). The membranes were blocked
in 5% milk in Tris-buffered saline with 0.1% Tween 20 (Sigma-Aldrich,
St. Louis, U.S.A.) (TBS-T, 137 mM NaCl, 20 mM Tris-base, pH 7.4, 0.1%
Tween 20) for 1 h and incubated with primary antibody overnight at
4 °C. As phospho-dependent antitau antibodies, we used: antihuman
DC217 (1:50, Axon Neuroscience R&D SE, Bratislava, Slovakia) and
monoclonal antihuman pThr212 (1:1000, Invitrogen Life Technologies,
Carlsbad, U.S.A.). For total tau, we used an antihuman DC190 antibody
(recognizing epitope 368–376, 1:50, Axon Neuroscience R&D
SE, Bratislava, Slovakia). Membranes were incubated with horseradish
peroxidase (HRP)-conjugated secondary antibody in TBS-T (1:3000, Dako,
Glostrup, Denmark) for 1 h at RT. Immunoreactive proteins were detected
by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate,
Thermo Scientific, Pittsburgh, U.S.A.) and the signals were digitized
by Image Reader LAS-3000 (FUJIFILM, Bratislava, Slovakia) (Figure S1 of the Supporting Information , SI ).
into 12% SDS-polyacrylamide gels and transferred to a nitrocellulose
membrane in 10 mM N-cyclohexyl-3-aminopropanesulfonic
acid (CAPS, pH 11, Roth, Karlsruhe, Germany). The membranes were blocked
in 5% milk in Tris-buffered saline with 0.1% Tween 20 (Sigma-Aldrich,
St. Louis, U.S.A.) (TBS-T, 137 mM NaCl, 20 mM Tris-base, pH 7.4, 0.1%
Tween 20) for 1 h and incubated with primary antibody overnight at
4 °C. As phospho-dependent antitau antibodies, we used: antihuman
DC217 (1:50, Axon Neuroscience R&D SE, Bratislava, Slovakia) and
monoclonal antihuman pThr212 (1:1000, Invitrogen Life Technologies,
Carlsbad, U.S.A.). For total tau, we used an antihuman DC190 antibody
(recognizing epitope 368–376, 1:50, Axon Neuroscience R&D
SE, Bratislava, Slovakia). Membranes were incubated with horseradish
peroxidase (HRP)-conjugated secondary antibody in TBS-T (1:3000, Dako,
Glostrup, Denmark) for 1 h at RT. Immunoreactive proteins were detected
by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate,
Thermo Scientific, Pittsburgh, U.S.A.) and the signals were digitized
by Image Reader LAS-3000 (FUJIFILM, Bratislava, Slovakia) (
5
Western Blot Analysis of LC3 Protein
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Cells were harvested in LIPA buffer (Sigma) as previously reported13 (link). After centrifugation at 15,000 × g for 5 min, the supernatant was boiled for 5 min in SDS-PAGE sample buffer (Wako) and loaded onto 5–20% gradient or 15% gels for 1~2 h. Proteins were transferred to nitrocellulose membranes (Bio-Rad) by using the Trans-Blot Electrophoretic Transfer cell (Bio-Rad). The membrane was washed in 20 mM Tris-HCl buffer with 1% Tween 20 (TBS-T) (Sigma-Aldrich), incubated for 1 h in blocking buffer (5% milk powder (Merck) in TBS-T), and then further incubated overnight with primary antibodies; Anti-LC3 (Cell signaling) and anti-GAPDH (Santa Cruz) antibodies were purchased and diluted in blocking buffer by 1:3000. The horseradish peroxidase (HRP)-labeled secondary antibody (Dako) was diluted by 1:5000 in TBS-T for secondary antibody staining for 1 h at RT. The membrane was then washed in TBS-T and the signals were developed using the Lumi-Light reagent (Roche) for 5 min. The signal was detected on the LAS-3000 imaging system (Fuji).
6
Immunofluorescence Analysis of Mouse Lymph Nodes
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Cryostat sections of mLNs were fixed in acetone/methanol solution (1:1, 10 min, −20 °C) and subjected to immunofluorescence histochemical analysis according to standard protocols. Briefly, the sections were rehydrated in TBST (0.1 M Tris pH 7.5, 0.15 M NaCl, and 0.1% Tween-20), preincubated with TBST containing 5% swine serum (Dako, Hamburg, Germany) and stained with antibodies against B220 and CD11b (BD Biosciences, Franklin Lakes, NJ, USA), CXCL13 (R&D Systems, Minneapolis, MN, USA), ERTR-7 (BMA, Augst, Switzerland), FDC-M1 (ImmunoKontact, Frankfurt, Germany), CD31-APC (BioLegend, San Diego, CA, USA) and Lyve-1 (kindly provided by R. Förster) in 2.5% serum/TBST. The unconjugated antibodies were then visualized using goat anti-rat Cy5 (Invitrogen, Carlsbad, CA, USA) or anti-rabbit Cy3 (Jackson ImmunoResearch, West Grove, PA, USA). The nuclei were visualized by DAPI staining (1 μg/ml DAPI/TBST), and the sections were mounted with Fluorescent Mounting Medium (Dako, Hamburg, Germany). Images were acquired using a Zeiss Axioskop 40 microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) connected to an AxioCam MRm (Carl Zeiss, Göttingen, Germany).
7
Immunohistochemical Analysis of Cadherin Expression
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The mouse subcutaneous heterotransplants were fixed in 10% neutral buffered formalin for 16–18 hours. All tissues were transferred to 70% ethanol and dehydrated in 100% ethanol. Dehydrated tissues were cleared in xylene, infiltrated, and embedded in paraffin. Tissue sections were cut at 3–5 μm for use in immuno-histochemical protocols. Prior to immuno-staining, sections were immersed in preheated citrate buffer pH 6.0 and heated in a steamer for 20 minutes. The sections were allowed to cool to room temperature and immersed into TBS-T (Dako, Carpinteria, CA) for 5 minutes. The immuno-staining was performed using the Dako ARKTM (Animal Research Kit, K3954, Dako, Carpinteria, CA) following the manufacturer’s recommended protocol. The N-cadherin antibody (Life Technology) and the E-cadherin antibody (Santa Cruz Biotechnology) were used at a dilution of 1:100. Liquid diaminobenzidine was used for visualization. Slides were rinsed in distilled water, dehydrated in graded ethanol, cleared in xylene, and coverslipped. Normal human kidney and Dako Universal Negative were used as positive and negative controls.
8
Lysenin-Based Smooth Muscle Staining
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Epon sections were hydrated in tris buffered saline (TBS) (Dako) with 0.1% Tween-20 (TBS-T; Dako) for 5 minutes. For SM staining, a 5 μg/mL solution of lysenin protein (Peptides International, Louisville, KY) was diluted in TBS with 0.5% BSA (TBS-B; Dako), placed onto the slides and incubated at 37°C for 1 hour. After rinsing in TBS-T, a 1:2500 dilution of rabbit antilysenin antiserum (Peptides International) diluted in TBS-B was added to the slides for 30 minutes at RT. After another TBS-T rinse, MACH2 rabbit alkaline phosphatase polymer (Biocare Medical, Concord, CA) was applied for 30 minutes at RT. After rinsing in TBS-T, slides were developed in Vulcan Fast Red chromagen (Biocare Medical) and counterstained in a 1:10 dilution of Richardson stain (heated to 60°C) for 15 seconds. Once thoroughly rinsed in running tap water, the slides were air dried, mounted with TBS SHUR/Mount Xylene-Based Mounting Medium (Triangle Biomedical Systems Inc.), and coverslipped. Using this method, SM appeared red against a light blue background, rendering the sections suitable for MetaMorph image analysis. In cases with very low levels of SM (eg, patient 3) MetaMorph was performed on lysenin-stained sections, in which the red/blue color separation was more distinct than the purple/blue color separation of the direct method described above.
9
Immunofluorescence Staining of Snail Knockdown
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5×103 cells were plated into 16 well chamber slides (Bio-Tek, Nunc, Winooski, VT). For treatments, cells were either untreated, treated with control siRNA or Snail siRNA. Fixation was performed with methanol/ethanol 1∶1 volume for 5 min, followed by washes with 1× PBS and blocking with protein blocking solution without serum (Dako, Camarillo, CA) for 10 min at room temp. Subsequently, slides were incubated with primary antibody at 1∶50 or 1∶100 dilutions in Dako antibody diluent solution for 1 h at room temp. Slides were washed with 1× TBS-T (Dako, Camarillo, CA), then incubated with secondary antibody in the dark for 1 h at room temp. Secondary antibodies used were anti-rabbit Oregon green 488, anti-mouse Alexa red 594 (Invitrogen, Carlsbad, CA) or anti-goat Texas red (Vector Laboratories, Burlingame, CA). Slides were washed with 1× TBS-T and double deionized water, prior to counterstaining with DAPI (1 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA). Slides were mounted using Fluorogel mounting medium (Electron Microscopy Sciences, Hatfield, PA). Fluorescence microscopy was performed using Zeiss microscope and Axiovision Rel 4.8 software.
10
Immunofluorescence Staining of Cell Lines
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5×103 cells were plated into 16 well chamber slides (Bio-Tek, Nunc, Winooski, VT). For treatments, cells were either untreated, treated with 5 or 20 μm Z-FY-CHO for 3 days. Fixation was performed with methanol/ethanol 1:1 volume for 5 min, followed by washes with 1× PBS and blocking with protein blocking solution without serum (Dako, Camarillo, CA) for 10 min at room temp. Subsequently, slides were incubated with primary antibody at 1 1:100 dilution in Dako antibody diluent solution for 1 hr. at room temp. Slides were washed with 1× TBS-T (Dako, Camarillo, CA), then incubated with secondary antibody in the dark for 1 hr. at room temp. Secondary antibodies used were anti-rabbit Oregon green 488 or anti-mouse Alexa red 594 (Invitrogen, Carlsbad, CA). Slides were washed with 1× TBS-T and double deionized water, prior to counterstaining with DAPI (1 μg/ml, Santa Cruz Biotechnology, Santa Cruz, CA). Slides were mounted using Fluorogel mounting medium (Electron Microscopy Sciences, Hatfield, PA). Fluorescence microscopy was performed using Zeiss microscope and Axiovision Rel 4.8 software.
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