PMC:7432599 / 44442-46387
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7432599","sourcedb":"PMC","sourceid":"7432599","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7432599","text":"4.12. Immunohistochemistry of UCP-1 in Brown and White Adipose Tissues and Macrophages in Subcutaneous Adipose Tissue\nThe adaptive thermogenesis marker uncoupling protein one (UCP-1) and macrophage marker F4/80 were determined in 4 μm thick sections of formalin-fixed paraffin embedded tissue according to Leal-Díaz et al. [63] and Méndez-Flores et al. [65] with some modifications. Endogenous peroxidase was blocked with 3% H2O2 solution. Then, non-specific background staining was avoided with the immunohistochemistry background blocker (Enzo Life Sciences Inc., Farmingdale, NY, USA). Brown and white adipose tissues were incubated with rabbit monoclonal anti-mouse UCP-1 (Abcam, Cambridge, MA, USA) and subcutaneous adipose tissue was incubated with F4/80 IgG2a,k antibody (Santa Cruz Biotechnology, Dallas, TX, USA) diluted at 10 µg/mL for 40 min at room temperature. Binding was identified with Universal Dako labelled streptavidin biotin reagent + peroxidase for primary antibodies from rabbit, mouse and goat (Dako, Glostrup, Denmark). Slides were incubated with streptavidin peroxidase for 15 min, followed by incubation with the peroxidase substrate 3,3′-diaminobenzidine (DAB; SIGMA-Aldrich) for 10 min. The sections were counterstained with hematoxylin, dehydrated with alcohol and xylene, and mounted in resin. Instead of primary antibody, negative controls were performed with normal human serum diluted 1:100 and with the IHC universal negative control reagent specifically designed to work with rabbit, mouse, and goat antibodies (IHC universal negative control reagent, Enzo Life Sciences, Inc.). Instead of the primary antibody the reactive blank was incubated with saline-egg albumin (Sigma-Aldrich). Controls excluded nonspecific staining or endogenous enzymatic activities. We examined at least two different sections of each tissue. Digital photographs were taken from each section at 40X magnification as described above.","divisions":[{"label":"title","span":{"begin":0,"end":117}}],"tracks":[{"project":"2_test","denotations":[{"id":"32752280-27678062-52426044","span":{"begin":324,"end":326},"obj":"27678062"},{"id":"T22489","span":{"begin":324,"end":326},"obj":"27678062"}],"attributes":[{"subj":"32752280-27678062-52426044","pred":"source","obj":"2_test"},{"subj":"T22489","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec9b93","default":true}]}]}}