Paraffin-embedded muscle sections were deparaffinized using xylene (Junsei, Tokyo, Japan), rehydrated using an ethanol gradient, and treated with methanol/H2O2 (Junsei) to quench endogenous peroxidase activity. Muscle sections were blocked with 1% normal goat serum (SeraCare Life Sciences, Milford, MA, USA) and incubated with protein-specific antibody [FMOD, MSTN, MYOG, MuRF1, Atrogin1, RAGE, Glb1, PPARγ, CD36, and CD163 (1:50)] overnight at 4 °C. Sections were then treated with HRP-conjugated secondary antibody (1:100; Santa Cruz Biotechnology), incubated for 1 h at room temperature, counterstained with hematoxylin, dehydrated, mounted, and examined under an optical microscope (Leica, Seoul, Korea). Morphological changes were examined in hematoxylin and eosin-stained sections under an optical microscope (Leica, Wetzlar, Germany).
Normal goat serum (ngs)
Manufactured by LGC
Sourced in United States
Normal goat serum is a laboratory-grade biological product derived from the blood of healthy goats. It is commonly used as a blocking agent or diluent in various immunological and biochemical assays to reduce non-specific binding and background signals.
Automatically generated - may contain errors
Lab products found in correlation
Xylene, by Junsei (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Optical microscope, by Leica (1 mentions)
Methanol h2o2, by Junsei (1 mentions)
Citrate buffer, by Merck Group (1 mentions)
Acrymount, by StatLab (1 mentions)
Ez retriever system, by BioGenex (1 mentions)
Horseradish peroxidase conjugated streptavidin, by Vector Laboratories (1 mentions)
Ethanol, by Merck Group (1 mentions)
Hematoxylin and eosin, by Thermo Fisher Scientific (1 mentions)
Hematoxylin, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 protocols using normal goat serum (ngs)
1
Immunohistochemical Analysis of Muscle Proteins
2
Immunohistochemical Profiling of Tissue Samples
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Paraformaldehyde-fixed, paraffin-embedded tissue sections were deparaffinized, rehydrated, and then boiled using the EZ-Retriever System (BioGenex) with 0.01 mol/liter citrate buffer, pH 6.0 (Sigma-Aldrich), for antigen retrieval. Endogenous peroxidases were blocked with 0.3% H2O2 for 15 min. Nonspecific epitopes were blocked with 10% normal goat serum (Seracare Life Sciences) for 30 min. The sections were incubated overnight at 4°C with antibodies against mouse CD8a, Gzmb, Ly6G, cleaved caspase 3 and Ki-6, and human CD15 and RORγt. Antibody details, including final concentrations, can be found in Table S3 . This was followed by using a SignalStain Boost IHC Detection Reagent and DAB Substrate Kit (Cell Signaling Technology) following the manufacturer’s instructions. Slides were then counterstained with hematoxylin, mounted in Acrymount (StatLab), and visualized under a light microscope.
3
Immunohistochemical Analysis of Muscle Tissues
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The sections of paraffin-embedded muscle tissue were deparaffinized and hydrated with xylene (Junsei, Tokyo, Japan) and ethanol (Merck), respectively, and endogenous peroxidase activity was quenched in 0.3% H2O2/methanol. The sections were then either stained with hematoxylin and eosin (Thermo Fisher Scientific) for morphological observation or blocked with 1% normal goat serum (SeraCare Life Sciences), incubated with primary antibodies [TTR (1:50), D2 (1:50), or FNDC5 (1:50)] overnight at 4 °C, and then incubated with horse radish peroxidase–conjugated secondary antibody (1:100). Positive signals were visualized by adding horse radish peroxidase-conjugated streptavidin (Vector, CA, USA). Nuclei of stained sections were stained with hematoxylin and then dehydrated, mounted, and observed by a light microscope (Leica, Wetzlar, Germany).
4
Immunofluorescence Staining of Cellular Proteins
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The cells were fixed with 4% formaldehyde (Sigma Aldrich) and permeabilized with 0.2% Triton X 100 (Sigma Aldrich). After blocking with 1% normal goat serum (SeraCare Life Sciences, Milford, MA, USA) for 30 min in a humid environment, cells were incubated with primary antibodies [TTR (1:50), MYOD (1:50), MYOG (1:50), MYL2 (1:50), D2 (1:50), RXRγ (1:50), TRα (1:50), or FNDC5 (1:50)] at 4 °C in a humid environment overnight. Secondary antibody (1: 100; Alexa Fluor 594 goat anti-rabbit or anti-mouse; Thermo Fisher Scientific) was applied for 1 h at room temperature. DAPI was used to stain the cells (Sigma-Aldrich) and imaged using a fluorescence microscope equipped with a digital camera (Nikon, Tokyo, Japan).
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