E-WAT from mice was perfused and fixed in 10% formalin saline, and then paraffin embedded. Paraffin-embedded sections were cut to 4 μM and the slides stained with hematoxylin and eosin. For uncoupling protein 1 (UCP1) staining, slides were deparaffinized, and antigen retrieval was carried out by heating sections to 95°C in sodium citrate buffer. Sections were blocked with goat serum before incubation with rabbit anti-UCP1 antibody (ab10983; Abcam Inc., Cambridge, United Kingdom) diluted 1:100. Following peroxidase blocking, horseradish peroxidase-conjugated goat anti-rabbit (P0448; Dako, Glostrup, Denmark) was used as the secondary antibody, and sections were incubated at 1:1000 in PBS for 1 h at room temperature. For staining, diaminobenzidine chromagen (K3468; Dako) was used according to the manufacturer’s instructions, and Mayer’s hematoxylin was used to counterstain.
Horseradish peroxidase conjugated goat anti rabbit p0448
Manufactured by Agilent Technologies
Sourced in Denmark
Horseradish peroxidase-conjugated goat anti-rabbit (P0448) is an immunological reagent. It consists of goat-derived antibodies that specifically bind to rabbit immunoglobulins, with horseradish peroxidase conjugated to the antibodies.
Automatically generated - may contain errors
2 protocols using horseradish peroxidase conjugated goat anti rabbit p0448
1
Immunohistochemical Analysis of UCP1 in Mouse Adipose
2
Quantifying Angiogenic and Oxidative Factors
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VEGF protein in the muscle lysates were determined by electrochemiluminescence assay kit for VEGF 165 (Meso Scale Diagnostics, Maryland, USA) according to the manufacturer´s guidelines. The values were normalized to the total protein content in each sample. eNOS, SOD-2 and NADPH oxidase analysis Lysate proteins were separated using SDS gels (Bio-Rad Laboratories) and transferred (semidry) to PVDF membranes (Immobilon Transfer Membrane, Millipore). The membranes were incubated with primary antibodies to eNOS (610297 BD Transduction Laboratories), SOD-2 (#06-984, Millipore, Billerica, US), NADPH oxidase (#610912, BD transduction, San Jose, Ca, USA) or GAPDH (ab9484, Abcam, Cambridge, UK). Secondary antibody horseradish-peroxidase-conjugated goat anti-rabbit (P-0448, Dako, Glostrup, Denmark) or horseradish-peroxidase-conjugated goat anti mouse (Jackson ImmunoResearch, USA) was used for detection of the proteins. Subsequent to exposure (Kodak Image Station, 2000MM) and quantification (Kodak Molecular Imaging software), the protein content was expressed in arbitrary units related to human standards.
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