For the detection of GATA3, a monoclonal rabbit antibody (Cell Signalling Technologies) was used. FLG was stained with a monoclonal mouse antibody purchased from Abcam and for FLG2detection, a polyclonal rabbit antibody (Sigma-Aldrich) was used. For all three targets the Envision+ kits from Dako (Hamburg, Germany) were used (FLG: HRP.Mouse (AEC+); GATA3: HRP.Rabbit (DAB+); FLG2: HRP.Rabbit AEC+)). The detection of Lucifer Yellow was performed with a polyclonal rabbit antibody (bought from ThermoScientific) in combination with HRP-Green Solution Set (42 life sciences GmbH & Co. KG, Bremerhaven, Germany). In all experiments the corresponding isotype control was added as a control. For the staining of FLG, GATA3, and Lucifer Yellow the “Target Retrieval Solution, ph9” and for FLG2 the “Target Retrieval Solution, ph6” (both Dako) was used. An Axio Scan.Z1 (Zeiss, Jena, Germany) was used for digitalization of the immunohistological specimens depicted in this study. Quantification of the staining intensity was done by using the software cellSense Dimension with Count & Measure Solution (Olympus Deutschland GmbH, Hamburg, Germany).
Monoclonal mouse antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Monoclonal mouse antibody is an immunoglobulin protein produced by a single clone of cells. It is derived from a mouse host and recognizes a specific target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Monoclonal rabbit antibody, by Cell Signaling Technology (1 mentions)
Target retrieval solution ph 6, by Agilent Technologies (1 mentions)
Target retrieval solution ph 9, by Agilent Technologies (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
3 protocols using monoclonal mouse antibody
1
Immunohistochemical Staining Protocol
2
Quantitative Western Blot Analysis of SERCA2a and Phospholamban
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Western blotting was performed as previously described (18 (link)). Proteins from homogenized left ventricles were separated by
10% or 15% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, which
were incubated with mouse monoclonal antibodies for SERCA-2a (1:1000, Affinity
BioReagents, USA), phospholamban (PLB; 0.5 µg/mL, Affinity BioReagents), and PLB
phosphorylated at serine 16 (pPLBSer16 1:5000, Badrilla, UK). After they
were washed, the membranes were incubated with anti-mouse (1:5000, Stressgen, Canada)
or anti-rabbit (1:7000, Stressgen) immunoglobulin antibodies conjugated to
horseradish peroxidase. After they were thoroughly washed, immunocomplexes were
detected using an enhanced horseradish peroxidase/luminol chemiluminescence system
(ECL plus, Amersham International, UK) and film (Hyperfilm ECL International).
Signals on the immunoblot were quantified with the Image J computer program (NIH,
USA). Each membrane was reprobed to determine GAPDH expression using a monoclonal
mouse antibody (1:5000, Abcam Cambridge, USA).
10% or 15% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, which
were incubated with mouse monoclonal antibodies for SERCA-2a (1:1000, Affinity
BioReagents, USA), phospholamban (PLB; 0.5 µg/mL, Affinity BioReagents), and PLB
phosphorylated at serine 16 (pPLBSer16 1:5000, Badrilla, UK). After they
were washed, the membranes were incubated with anti-mouse (1:5000, Stressgen, Canada)
or anti-rabbit (1:7000, Stressgen) immunoglobulin antibodies conjugated to
horseradish peroxidase. After they were thoroughly washed, immunocomplexes were
detected using an enhanced horseradish peroxidase/luminol chemiluminescence system
(ECL plus, Amersham International, UK) and film (Hyperfilm ECL International).
Signals on the immunoblot were quantified with the Image J computer program (NIH,
USA). Each membrane was reprobed to determine GAPDH expression using a monoclonal
mouse antibody (1:5000, Abcam Cambridge, USA).
3
Surfactant-Bound Protein Detection
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Surfactant-bound proteins were detected using an adapted previously described protocol.56 (link) Nanoparticles were incubated in concentrated BAL as before. Samples were then centrifuged at 5000g for 10 min to separate NPs and bound components from the unbound solution. The supernatant was aspirated and saved, while the pellet was washed in PBS and centrifuged three times to remove any unbound proteins. NuPAGE LDS sample buffer (Invitrogen, UK) was then added to the saved supernatant and resuspended pellet, and the samples were heated at 80 °C for 10 min. Samples were then loaded into 4–12% NuPAGE Bis-Tris gels. Gels were run in NuPAGE MOPS SDS running buffer, and protein bands were then transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were then washed and blocked for 1 h in TBST containing 5% BSA. Membranes were probed for SP-A using a polyclonal rabbit antibody (1 μg/mL; Abcam, UK) or SP-D using a monoclonal mouse antibody (0.3 μg/mL, Abcam).
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