The SN was isolated from mice 12 weeks after AAV injection using a dissecting microscope. Tissues were lysed in lysis buffer containing 5 M NaCl, 10% NP40, 1 M Tris-HCl (pH 7.4), 50% glycerol, and cOmplete protease inhibitor cocktail (Roche Applied Science). Each sample was heated in 6× loading buffer at 95 °C for 5 min, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (Millipore). Membranes were treated with blocking solution (1% milk in 1× phosphate-buffered saline with 0.1% Tween 20®) for 30 min and incubated with rabbit anti-RGMa (1:100, 28045, Immuno-Biological Laboratories) and rabbit anti-β-actin (1:1000, 4970; Cell Signaling Technology) overnight at 4 °C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody against rabbit IgG (1:1000; Cell Signaling Technology) for 1 h in RT. Detection was performed using western blotting Substrate Plus (Thermo Fisher Scientific).
Horseradish peroxidase conjugated secondary antibody against rabbit igg
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated secondary antibody against rabbit IgG is a laboratory reagent used in immunoassays and other applications. It consists of a secondary antibody that recognizes and binds to rabbit immunoglobulin G (IgG) molecules, and is conjugated to the enzyme horseradish peroxidase. This enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of target proteins in samples.
Automatically generated - may contain errors
Lab products found in correlation
Biological microscope bx41, by Olympus (2 mentions)
Dp72 color digital camera, by Olympus (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions)
Formalin solution, by Merck Group (1 mentions)
Hematoxylin eosin, by Merck Group (1 mentions)
Antibody against ki67, by Abcam (1 mentions)
Sigmafast dab, by Merck Group (1 mentions)
3 protocols using horseradish peroxidase conjugated secondary antibody against rabbit igg
1
Western Blot Analysis of RGMa Expression
2
Immunohistochemical Analysis of Liver FGF21
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Liver specimens collected before or 2 hours post-implantation were fixed in 10% formalin solution (Sigma-Aldrich, USA); and paraffin-embedded liver sections were stained with hematoxylin-eosin (Sigma-Aldrich, USA). For FGF21 immunohistochemical staining, deparaffinized and dehydrated liver sections were incubated with an affinity-purified rabbit antibody against FGF21 (Antibody and Immunoassay Services, Hong Kong, China) in a solution containing 3% bovine serum albumin overnight at 4 °C, followed by the reaction with a horseradish peroxidase-conjugated secondary antibody against rabbit IgG (Cell Signaling Technology, USA) and 3,3′-Diaminobenzidine tetrahydrochloride (Sigma-Aldrich, USA). Immuno-stained slides were visualized with an Olympus biological microscope BX41, and images were captured with an Olympus DP72 color digital camera.
3
Immunohistochemical and ELISA analysis of adiponectin
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Adipose tissues were fixed in 10% formalin solution for 24 hr, embedded in paraffin, and sectioned at 5 mm. Deparaffinized and dehydrated sections were incubated with affinity-purified rabbit antibody against mouse adiponectin (AIS, HKU), rabbit antibody against mouse UCP1 (Abcam) in PBS containing 3% BSA overnight at 4 C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody against rabbit IgG (Cell Signaling Technology), and developed by SIGMAFAST DAB (Sigma). For immunocytochemical staining, bone marrow-derived macrophages were fixed in ice-cold 4% formaldehyde for 10 min. The cells were then incubated with antibody against Ki67 (Abcam) or adiponectin in PBS containing 3% BSA overnight at 4 C, washed and incubated with fluorochrome-conjugated secondary antibodies (Alexa Fluor 555 anti-rabbit, Molecular Probes), and diluted in PBS for 1 hr at room temperature at dark. Slides were counterstained with DAPI. Tissue sections and cell slides were visualized with an Olympus biological microscope BX41, and images were captured with an Olympus DP72 color digital camera. Adiponectin levels in serum were measured by mouse adiponectin ELISA kit (AIS, HKU).
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