mouse primary antibodies for ucp1  (Abcam)

Journal: Neuroscience

Article Title: Diminished Cold Avoidance Behaviours after Chronic Cold Exposure - Potential Involvement of TRPM8.

doi: 10.1016/j.neuroscience.2021.06.014

Figure Lengend Snippet: Fig. 1. Schematic summary of experiments. Male C57BL/6 mice were acclimated for four weeks to appropriate thermal conditions in two separate climate chambers by gradually lowering the ambient temperature (Ta) to 6 C (Cold group; indicated by blue), or increasing to thermoneutral temperature 27 C (Thermoneutral group; indicated by red). After acclimation, mice were assigned to three different experiments as indicated: First, thermosensory preference behaviour testing using Two Plate Choice Test and Thermal Gradient Test. Second, injections with either TRPM8 agonist icilin or vehicle prior to Thermal Gradient Test, and third, Evaluation of cold acclimation responses including thermography imaging and molecular analyses of TRPM8 and UCP1 expression in dorsal root ganglion (DRG) and interscapular brown adipose tissue (iBAT) homogenates, respectively.

Article Snippet: Then the membranes were incubated at 4 C over night with a fluorescent antibody against TRPM8 (REF#NBP1-97311C; TRPM8 monoclonal antibody conjugated with Dylight 650 fluorochrome, Novus Biologicals, USA) diluted 1:2000 in Immobilon Block-FL, or with a primary mouse antibody against UCP1 (REF # MAB6158, R&D Systems) diluted 1:1000 in Immobilon Block-FL, which was detected using a 1:5000 dilution of fluorescent Goat anti-Mouse secondary antibody (Alexa Fluor 647, # A32728, Thermo Fisher Scientific, USA).

Techniques: Imaging, Expressing

Journal: Scientific Reports

Article Title: A novel peptide that improves metabolic parameters without adverse central nervous system effects

doi: 10.1038/s41598-017-13690-9

Figure Lengend Snippet: Quantitative analyses in inguinal adipose tissue of DIO rats. Panels a–d: Immunohistochemical representative images (400x magnification, scale bar = 50 µm) showing specific UCP1-immunostained cells from the inguinal white adipose tissue of DIO Wistar rats (oral treatments: panel a, saline; panel b, Pep19, 100 μg/Kg; panel c, Pep19, 300 μg/Kg; panel d, Pep19, 600 μg/Kg). Quantitative analyses of UCP1 labeled cells were determined from at least 25 different fields in each slice (n = 8–10), and examined using ImageJ software at 400x magnification; results are expressed as number of UCP1 positive cells per mm 2 . Representative UCP1-immunostained cells (fast red dye) obtained from each treatment are indicated by arrows. Panel e: Quantitative analyses suggest that oral administration of Pep19 600 μg/Kg increases the number of UCP1-immunostained cells in the inguinal adipose tissue compared to saline or other treatments (n = 8–10; *p < 0.05, saline vs Pep19 600 μg/Kg). Note that in Pep19 treated animals (panels, c and d) immunostained-UCP1 cells seem to occur in fibrotic-like areas of the adipose tissue. Panel f: Quantitative Western blot analysis suggested similar levels of UCP1 expression in either saline or Pep19 (600 μg/Kg) treated groups of DIO animals; (each line shown on the upper panel f, is representative of one individual animal treated with either saline or Pep19 600 μg/Kg; n = 7). Note that the ratio of UCP1 negative cells to positive cells is fairly large and this could contribute to lack in change in the protein levels seen in the Western Blots (panel f). Panel g: Quantitative analyses of the number of adipocytes (adipocytes/µm 2 ; 500–800 cells were counted per animal (n = 3), in 14–18 different fields of the H&E stained slices of adipose tissue from animals treated with either saline or Pep19 (600 μg/Kg; *p < 0.05). Panel h: Quantitative analyses of the adipocyte area were performed measuring 15 different adipocytes in each of the 6 fields analyzed per H&E stained slices from each animal (n = 3), using H&E stained slices of adipose tissue from animals treated with either saline or Pep19 (600 μg/Kg; *p < 0.01). All results are expressed as the means ± standard error of the mean (SEM). The statistical comparisons were performed using Student’s t-test or analysis of variance (ANOVA), followed by ad-hoc Tukey’s test using GraphPad Prism software. Crude Western blot membranes are shown on Supplemental material.

Article Snippet: Membranes were blocked for 1 h at room temperature in Tris-buffered saline Tween-20 buffer with 5% nonfat dry milk, followed by overnight incubation at 4 °C with specific mouse primary antibodies for UCP1 (Abcam, ab23841) or GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:1000 in Tris-buffered saline with 5% nonfat dry milk.

Techniques: Immunohistochemical staining, Labeling, Software, Western Blot, Expressing, Staining

Journal: Scientific Reports

Article Title: A novel peptide that improves metabolic parameters without adverse central nervous system effects

doi: 10.1038/s41598-017-13690-9

Figure Lengend Snippet: Signaling pathways induced by Pep19 in 3T3-L1 adipocyte cells. Panels a and b: Relative UCP1 expression in 3T3-L1 adipocyte cells. Panel a: Cells were exposed to rosiglitazone (RSG, 5µM), or different concentrations of hemopressin (HP, 0.1–10 µM) or Pep19 (0.1–10 µM). Panel b: 3T3-L1 adipocyte cells exposed to Pep19 (1 µM) for 24 h in the absence or presence of either RSG (5 µM), the CB1R agonist WIN55,212–2 (1 µM), the CB1R antagonist AM251 (1 µM) or the CB1R inverse agonist HP (1 µM). Western blots were conducted using mouse anti-UCP1 antibodies, and anti-GAPDH antibodies were used as loading controls. Panels c and d: 3T3-L1 cells were starved for 16 h in serum-free medium prior to stimulation (vehicle or Pep19, 1 μM) for the indicated time period. Western blots were carried out using: Panel c, mouse monoclonal anti-phosphoERK1/2, and rabbit polyclonal anti-total ERK1/2; Panel d, rabbit phospho-AKT S473 (anti-pAKT, S473) and mouse monoclonal anti-tubulin antibodies. Imaging and band intensity measurements were performed using the Odyssey imaging system (LI-COR, Lincoln, NE) according to the manufacturer’s protocols. Data are representative of three independent experiments that produced similar results. Unt, cells not treated with vehicle (Veh) or peptide 19 (Pep19). The statistical comparisons were performed using Student’s t-test or analysis of variance (ANOVA), followed by ad-hoc Tukey’s test using GraphPad Prism software * p < 0.05; ** p < 0.001. Crude Western blot membranes are shown on Supplemental material.

Article Snippet: Membranes were blocked for 1 h at room temperature in Tris-buffered saline Tween-20 buffer with 5% nonfat dry milk, followed by overnight incubation at 4 °C with specific mouse primary antibodies for UCP1 (Abcam, ab23841) or GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:1000 in Tris-buffered saline with 5% nonfat dry milk.

Techniques: Expressing, Western Blot, Imaging, Produced, Software

Journal: Scientific Reports

Article Title: A novel peptide that improves metabolic parameters without adverse central nervous system effects

doi: 10.1038/s41598-017-13690-9

Figure Lengend Snippet: Quantitative analyses in inguinal adipose tissue of DIO rats. Panels a–d: Immunohistochemical representative images (400x magnification, scale bar = 50 µm) showing specific UCP1-immunostained cells from the inguinal white adipose tissue of DIO Wistar rats (oral treatments: panel a, saline; panel b, Pep19, 100 μg/Kg; panel c, Pep19, 300 μg/Kg; panel d, Pep19, 600 μg/Kg). Quantitative analyses of UCP1 labeled cells were determined from at least 25 different fields in each slice (n = 8–10), and examined using ImageJ software at 400x magnification; results are expressed as number of UCP1 positive cells per mm 2 . Representative UCP1-immunostained cells (fast red dye) obtained from each treatment are indicated by arrows. Panel e: Quantitative analyses suggest that oral administration of Pep19 600 μg/Kg increases the number of UCP1-immunostained cells in the inguinal adipose tissue compared to saline or other treatments (n = 8–10; *p < 0.05, saline vs Pep19 600 μg/Kg). Note that in Pep19 treated animals (panels, c and d) immunostained-UCP1 cells seem to occur in fibrotic-like areas of the adipose tissue. Panel f: Quantitative Western blot analysis suggested similar levels of UCP1 expression in either saline or Pep19 (600 μg/Kg) treated groups of DIO animals; (each line shown on the upper panel f, is representative of one individual animal treated with either saline or Pep19 600 μg/Kg; n = 7). Note that the ratio of UCP1 negative cells to positive cells is fairly large and this could contribute to lack in change in the protein levels seen in the Western Blots (panel f). Panel g: Quantitative analyses of the number of adipocytes (adipocytes/µm 2 ; 500–800 cells were counted per animal (n = 3), in 14–18 different fields of the H&E stained slices of adipose tissue from animals treated with either saline or Pep19 (600 μg/Kg; *p < 0.05). Panel h: Quantitative analyses of the adipocyte area were performed measuring 15 different adipocytes in each of the 6 fields analyzed per H&E stained slices from each animal (n = 3), using H&E stained slices of adipose tissue from animals treated with either saline or Pep19 (600 μg/Kg; *p < 0.01). All results are expressed as the means ± standard error of the mean (SEM). The statistical comparisons were performed using Student’s t-test or analysis of variance (ANOVA), followed by ad-hoc Tukey’s test using GraphPad Prism software. Crude Western blot membranes are shown on Supplemental material.

Article Snippet: Next, membranes were incubated overnight at 4 °C with specific mouse primary antibodies anti-UCP1 (Abcam, ab23841) or GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:1000 in Tris-HCl-buffered saline, pH 7.4, containning 5% nonfat dry milk.

Techniques: Immunohistochemical staining, Saline, Labeling, Software, Western Blot, Expressing, Staining

Journal: Scientific Reports

Article Title: A novel peptide that improves metabolic parameters without adverse central nervous system effects

doi: 10.1038/s41598-017-13690-9

Figure Lengend Snippet: Signaling pathways induced by Pep19 in 3T3-L1 adipocyte cells. Panels a and b: Relative UCP1 expression in 3T3-L1 adipocyte cells. Panel a: Cells were exposed to rosiglitazone (RSG, 5µM), or different concentrations of hemopressin (HP, 0.1–10 µM) or Pep19 (0.1–10 µM). Panel b: 3T3-L1 adipocyte cells exposed to Pep19 (1 µM) for 24 h in the absence or presence of either RSG (5 µM), the CB1R agonist WIN55,212–2 (1 µM), the CB1R antagonist AM251 (1 µM) or the CB1R inverse agonist HP (1 µM). Western blots were conducted using mouse anti-UCP1 antibodies, and anti-GAPDH antibodies were used as loading controls. Panels c and d: 3T3-L1 cells were starved for 16 h in serum-free medium prior to stimulation (vehicle or Pep19, 1 μM) for the indicated time period. Western blots were carried out using: Panel c, mouse monoclonal anti-phosphoERK1/2, and rabbit polyclonal anti-total ERK1/2; Panel d, rabbit phospho-AKT S473 (anti-pAKT, S473) and mouse monoclonal anti-tubulin antibodies. Imaging and band intensity measurements were performed using the Odyssey imaging system (LI-COR, Lincoln, NE) according to the manufacturer’s protocols. Data are representative of three independent experiments that produced similar results. Unt, cells not treated with vehicle (Veh) or peptide 19 (Pep19). The statistical comparisons were performed using Student’s t-test or analysis of variance (ANOVA), followed by ad-hoc Tukey’s test using GraphPad Prism software * p < 0.05; ** p < 0.001. Crude Western blot membranes are shown on Supplemental material.

Article Snippet: Next, membranes were incubated overnight at 4 °C with specific mouse primary antibodies anti-UCP1 (Abcam, ab23841) or GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:1000 in Tris-HCl-buffered saline, pH 7.4, containning 5% nonfat dry milk.

Techniques: Protein-Protein interactions, Expressing, Western Blot, Imaging, Produced, Software