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7 protocols using ucp11 a

1

Histochemical Analysis of Adipose Tissue

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Hematoxylin and eosin (H&E) staining was performed using paraffin sections as previously described [21 (link)]. Briefly, adipose tissues were fixed using 10% formalin (Sigma, USA) for 24 h and subjected to paraffin embedding. Subsequently, tissue blocks embedded in paraffin were sectioned into 5 μm thick slice. After deparaffinization, sections were stained using ClearView H&E Y alcoholic solution (BBC biochemical, USA). For immunostaining, deparaffinized sections of adipose tissue were incubated with anti-UCP1 antibody (1:400; UCP11-A, Alpha Diagnostic International, USA) at 4 °C overnight and then with goat anti-rabbit Alexa Flour 488 (1:500; Thermo Fisher, USA) as a secondary antibody for 1 h at room temperature. Nuclei were stained with DAPI (Sigma, USA). LSM800 confocal microscope (Zeiss, Germany) was used to acquire images and the data were analyzed with Zen software (version 3.0).
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2

Preparation and Analysis of Cellular Lysates

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Total cell lysates were prepared in a buffer containing 50 mM Tris-HCl (pH 7.8; Thermo Fisher Scientific), 137 mM NaCl (Thermo Fisher Scientific), 10 mM NaF (Thermo Fisher Scientific), 1 mM EDTA (Thermo Fisher Scientific), 1% Triton X-100 (Thermo Fisher Scientific), 10% glycerol (Thermo Fisher Scientific), and the protease inhibitor cocktail (Roche) through 3 freeze/thaw cycles. Tissue lysates were prepared by homogenizing in a buffer containing 50 mM Tris (pH 7.6; Thermo Fisher Scientific), 130 mM NaCl (Thermo Fisher Scientific), 5 mM NaF (Thermo Fisher Scientific), 25 mM β-glycerophosphoate (Thermo Fisher Scientific), 1 mM sodium orthovanadate (Thermo Fisher Scientific), 10% glycerol (Thermo Fisher Scientific), 1% Triton X-100 (Thermo Fisher Scientific), 1 mM dithiothreitol (Thermo Fisher Scientific), 1 mM phenylmethanesulfonyl fluoride (PMSF) (Thermo Fisher Scientific), and the protease inhibitor cocktail (Roche). After centrifugation (12,000g, 4°C for 10 minutes), tissue lysates were separated on SDS-polyacrylamide gel (SDS-PAGE) and analyzed using the following antibodies: rabbit anti-UCP1 (UCP11-A, Alpha Diagnostic), rabbit anti-HSP90 (sc-7947, Santa Cruz Biotechnology Inc.), rabbit anti–phospho-PKA substrate (9624, Cell Signaling Technology), rabbit anti-TH (ab112, Abcam), and mouse anti-tubulin (sc-32293, Santa Cruz Biotechnology Inc.).
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3

Immunohistochemical Analysis of Ucp1 Protein

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Adipose tissues were dissected and directly fixed overnight in 10% formalin before paraffin embedding and H&E (hematoxylin and eosin) staining. Immunohistochemical analysis of Ucp1 protein was performed as described before49 (link). Briefly, unstained tissue sections were heated at 60 °C for 30 min before rehydration. After antigen retrieval by boiling the slides in 10 mM sodium citrate-citric acid solution (pH = 6.2) for 20 min, the tissue sections were incubated overnight at 4 °C with anti-Ucp1 antibody (UCP11-A, Alpha Diagnostic) prepared in blocking reagent (5% BSA, 0.5% Tween-20, 0.05% NaN3 in PBS). The slides were then incubated with ImmPRESS (peroxidase) polymer anti-rabbit IgG reagent, followed by exposure to 3,3-diaminobenzidine (Vector Lab). The slides were subsequently dehydrated for mounting (Permount, Thermo Fisher). Images were captured on Olympus BX51 microscope.
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4

Mitochondrial and Protein Expression Analysis

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Muscle homogenates were probed for proteins of mitochondrial oxidative phosphorylation (ab110413, Abcam), RyR (sc-13942, Santa Cruz), SERCA1 (ab2819, Abcam), SERCA2 (ab3625, Abcam), SLN (ABT13, Millipore), and GAPDH (2118, Cell Signaling). Brown adipose tissue (BAT), epidydymal white adipose (eWAT), or inguinal subcutaneous white adipose tissue (iWAT) were blotted for UCP1 (UCP11-A, Alpha Diagnostic). Additionally, BAT was blotted for COXIV (4844S, Cell Signaling) or Citrate Synthase (ab96600, Abcam).
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5

Mitochondrial and Protein Expression Analysis

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Muscle homogenates were probed for proteins of mitochondrial oxidative phosphorylation (ab110413, Abcam), RyR (sc-13942, Santa Cruz), SERCA1 (ab2819, Abcam), SERCA2 (ab3625, Abcam), SLN (ABT13, Millipore), and GAPDH (2118, Cell Signaling). Brown adipose tissue (BAT), epidydymal white adipose (eWAT), or inguinal subcutaneous white adipose tissue (iWAT) were blotted for UCP1 (UCP11-A, Alpha Diagnostic). Additionally, BAT was blotted for COXIV (4844S, Cell Signaling) or Citrate Synthase (ab96600, Abcam).
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6

IHC Analysis of UCP1 in Tissue

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Tissue sections were embedded in paraffin and stained with H&E. IHC analysis of UCP1 protein was performed as previously described (43 (link)). Briefly, unstained tissue sections were heated at 60°C for 30 minutes for rehydration. After antigen retrieval by boiling the slides in a 10 mM sodium citrate–citric acid solution (pH 6.2) for 20 minutes, the tissue sections were incubated overnight at 4°C with an anti-UCP1 antibody (UCP11-A, Alpha Diagnostic) prepared in blocking reagent (5% BSA [Thermo Fisher Scientific], 0.5% Tween-20 [Thermo Fisher Scientific], 0.05% NaN3 [MilliporeSigma] in PBS). The slides were then incubated with ImmPRESS (peroxidase) polymer anti–rabbit IgG (MP-7401-15, Vector Lab) reagent, followed by exposure to DAB (Vector Lab). After dehydration, the slides were mounted using Permount (Thermo Fisher Scientific), and images were captured using an Olympus BX51 microscope.
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7

Adipocyte Diameter Quantification and UCP1 Immunohistochemistry

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Hematoxylin and eosin (H&E) staining was conducted [22 (link)]. To measure the diameters of adipocytes, images obtained by 20× magnifying the H&E-stained paraffin sections were taken and used.
For immunohistochemistry, anti-UCP1 antibody (1:400 dilution; UCP11-A, Alpha Diagnostic International) was used to stain deparaffinized tissues at 4 °C overnight. Goat anti-rabbit Alexa Flour 488 (1:500 dilution; Thermo Fisher, Waltham, MA, USA) was used for a secondary antibody and stained for 1h at RT. DAPI (Sigma, St. Louis, MO, USA) was used to stain nuclei. Histological images were captured by LSM800 confocal microscope (Zeiss, Jena, Germany) and the adipocyte sizes were measured by Zen software (ver. 3.0).
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