Tissues were formalin-fixed, paraffin-embedded, sectioned and stained with haematoxylin and eosin (H&E) as described.21 (link) Immunohistochemistry and immunofluorescence were done as described.20 (link),21 (link) Following de-paraffinization, the immunostaining procedure of paraffin-embedded tissue sections was similar to that of frozen tissues and cultured cells. Primary antibodies: rabbit anti-UCP1 (1:200, Abcam, Cambridge, MA), rabbit anti-ERα (1:200, Abcam), rabbit anti-mouse β3-adrenergic-receptor (1:200, Abcam), mouse anti-PCNA (1:200, Millipore, Burlington, MA) and goat-anti-Perilipin (1:200, BD Biosciences, San Jose, CA). Secondary antibodies: Goat/Donkey anti- rabbit/mouse conjugated with AlexaFluor-488/Cy3, and Donkey anti-goat conjugated with Cy5 (1:500, Jackson ImmunoResearch, West Grove, PA). DAPI (1:500, Fluka, Milwaukee, WI). Nile-red (1 µg/mL, Sigma). Bright-field and fluorescent images were collected on: Olympus inverted IX70 microscope, Olympus upright BX40 microscope, Leica inverted DMi8 microscope or Leica upright DM6 microscope. An independent researcher reviewed slides to confirm results.
Protocol detail
Immunohistochemical Analysis of Adipose Tissues
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Adrenergic receptor
Antibodies
Cultured cells
Dapi
Donkey
Embedded paraffin
Eosin
Formalin
Frozen
Goat
Immunofluorescence
Immunohistochemistry
Microscope
Mouse
Paraffin
Pcna
Perilipin
Rabbit
Tissues
Ucp1
Corresponding Organization :
Other organizations : The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- Tissue preparation (formalin-fixed, paraffin-embedded, sectioned and stained with haematoxylin and eosin (H&E))
- Immunohistochemistry and immunofluorescence procedures
- Primary antibodies used (rabbit anti-UCP1, rabbit anti-ERα, rabbit anti-mouse β3-adrenergic-receptor, mouse anti-PCNA, goat-anti-Perilipin)
- Secondary antibodies used (Goat/Donkey anti-rabbit/mouse conjugated with AlexaFluor-488/Cy3, Donkey anti-goat conjugated with Cy5)
- Expression and localization of UCP1, ERα, β3-adrenergic-receptor, PCNA, and Perilipin proteins
- Tissue preparation and staining procedures
- Imaging techniques (Olympus inverted IX70 microscope, Olympus upright BX40 microscope, Leica inverted DMi8 microscope, Leica upright DM6 microscope)
- Positive control: Independent researcher reviewed slides to confirm results
- Negative control: Not explicitly mentioned
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