Equal volumes of sample and sample buffer [0.125 mol Tris-HCl (pH 6.8), 4.0% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.004% bromophenol blue] were mixed and heated at 95°C for 5 min. For Western blot detection of chIL-13 using the anti–chIL-13 mAb, yeast- or E. coli–expressed recombinant chIL-13 (1 μg/lane) was resolved on a 15% SDS-PAGE gel followed by electroblotting onto a nitrocellulose membrane (Immobilon-P, Millipore, Bedford, MA). Similarly, the cross-reactivity of these mAb was determined using chIL-10 and chIFN-γ at a concentration of 1 μg/lane (all from Kingfisher Biotech, St. Paul, MN). Blocking of the membranes was performed using SuperBlock Blocking Buffer in PBS (Thermo Fisher Scientific, Waltham, MA) followed by washing with 1x PBS-T. The membranes were independently incubated with 5 anti–chIL-13 mAb (1.0 μg/mL). HRP-conjugated rabbit anti–mouse IgG secondary Ab (1/1,000 dilution) was used to determine the immunoreactivity, and the bands were detected using a Clarity Western ECL Substrate (Bio-Rad, Hercules, CA); the membranes were visualized using the ChemiDoc imaging system (Bio-Rad, Hercules, CA).
Superblock blocking buffer in pbs
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Superblock Blocking Buffer in PBS is a solution designed to block non-specific binding in immunoassays. It is a ready-to-use buffer that contains a proprietary blend of proteins and blocking agents to reduce background and improve signal-to-noise ratios.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti fibronectin, by Merck Group (2 mentions)
Immobilon p, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Cri nuance fx camera system, by PerkinElmer (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
Image analysis system, by Uvitec (1 mentions)
Luminata crescendo, by Merck Group (1 mentions)
Nucleospin rna protein, by Macherey-Nagel (1 mentions)
Hybond c nitrocellulose membranes, by Thermo Fisher Scientific (1 mentions)
Anti human αsma, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Rabbit anti collagen 1, by Novus Biologicals (1 mentions)
Bz x700 fluorescence microscope, by Keyence (1 mentions)
5 protocols using superblock blocking buffer in pbs
1
Western Blot Detection of Chicken IL-13
2
Fibronectin Immunofluorescence in Murine Eyes
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After completion of the IOP time course, mouse eyes were enucleated and fixed in 4% PFA overnight. Eyes were embedded in paraffin, cut into 5-μm sections, and transferred to glass slides. Deparaffinization was performed by washing two times with xylene, 100% ethanol, 95% ethanol, and 50% ethanol for 2 minutes each. Slides were then soaked in PBS for 5 minutes. Tissues were blocked using Superblock Blocking Buffer in PBS (Thermo Fisher Scientific) for 30 to 60 minutes. Rabbit anti-fibronectin (EMD Millipore) 1:1000 dilution was used to label FN, followed by Alexa Fluor–labeled anti-rabbit Ig (Life Technologies) 1:1000 dilution. Prolong Gold mounting medium containing DAPI (Invitrogen-Molecular Probes) was used to mount the slides and imaged using fluorescent microscope Nikon ECLIPSE Ti-U (Nikon Instruments, Inc., Melville, NY, USA) equipped with a CRi Nuance FX Camera System (Perkin-Elmer, Waltham, MA, USA). All images were taken at ×400 magnification; scale bar represents 50 μm.
3
Measuring Antigen-Specific IgA in Sera
4
Quantitative Protein Expression Analysis
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Proteins were extracted using NucleoSpin RNA/protein (Macherey-Nagel) and quantified by the Bicinchoninic acid method. For each experimental condition, 30 μg of proteins were separated by electrophoresis on Bis-Tris gel (GenScript Biothec, Leiden, Netherlands) and transferred onto Hybond-C-nitrocellulose membranes (Life Technologies Ltd. Paisley, UK).
After 1 h in blocking solution (SuperBlock Blocking buffer in PBS, Thermo Scientific), membranes were incubated overnight at 4 °C with the following primary antibodies: anti-human αSMA (dilution 1:300, Sigma-Aldrich), S100A4 (dilution 1:100, Santa Cruz Biotechnology, Dallas, USA), COL1 (dilution 1:600, Proteintech), and FN (dilution 1:2,000, Sigma-Aldrich). The membranes were subsequently incubated with (HRP)-conjugated secondary antibodies (dilution 1:2,000; Cell Signaling, MA, USA). To confirm similar loading of protein samples into the gels and the efficiency in the electrophoretic transfer, membranes were incubated with primary HRP-conjugated antibody to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1:2,000; Santa Cruz Biotechnology). Protein synthesis was detected using the enhanced chemiluminescence system (Luminata Crescendo, Millipore), and the densitometric analysis was performed by the UVITEC Image Analysis System (UVITEC, Cambridge, UK).
After 1 h in blocking solution (SuperBlock Blocking buffer in PBS, Thermo Scientific), membranes were incubated overnight at 4 °C with the following primary antibodies: anti-human αSMA (dilution 1:300, Sigma-Aldrich), S100A4 (dilution 1:100, Santa Cruz Biotechnology, Dallas, USA), COL1 (dilution 1:600, Proteintech), and FN (dilution 1:2,000, Sigma-Aldrich). The membranes were subsequently incubated with (HRP)-conjugated secondary antibodies (dilution 1:2,000; Cell Signaling, MA, USA). To confirm similar loading of protein samples into the gels and the efficiency in the electrophoretic transfer, membranes were incubated with primary HRP-conjugated antibody to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1:2,000; Santa Cruz Biotechnology). Protein synthesis was detected using the enhanced chemiluminescence system (Luminata Crescendo, Millipore), and the densitometric analysis was performed by the UVITEC Image Analysis System (UVITEC, Cambridge, UK).
5
Early TM Changes in Transgenic Mice
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To evaluate early changes in the TM, an initial cohort of mice were transduced with viral vectors, Ad5.Null (n = 7), Ad5.Cre (n = 15), and Ad5.TGFβ2 (n = 9), and harvested 11-days after injection. IOP was recorded at 10-days postinjection. Eyes were fixed in 4% PFA overnight, embedded in paraffin, cut into 5 μm sections, and transferred to glass slides. Deparaffinization was performed by washing two times with xylene, 100% ethanol, 95% ethanol, and 50% ethanol for 2 minutes each. Slides then were soaked in PBS for 5 minutes. Tissues were blocked using Superblock Blocking Buffer in PBS (Thermo Fisher Scientific) for 60 minutes. Rabbit anti-fibronectin (EMD Millipore) 1:500 dilution, rabbit anti-collagen-1 (Novus Biologicals, LL) 1:250 dilution, mouse anti-collagen-4 (Sigma-Aldrich Corp., St. Louis, MO, USA) 1:250 dilution, and mouse anti-BAMBI (Abnova; Walnut, CA, USA) 1:250 dilution, were used as primary antibodies, followed by Alexa-Fluor–labeled anti-rabbit or anti-mouse antibodies (Life Technologies, Carlsbad, CA, USA) 1:500 dilution. Prolong Gold mounting medium containing DAPI (Invitrogen-Molecular Probes) was used to mount the slides, and sections were imaged using the Keyence BZ-X700 fluorescence microscope (Keyence Corporation of America, Itasca, IL). All images were taken at ×100 magnification; scale bars represent 100 μm.
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