monoclonal rabbit anti mouse antibody against perilipin  (Cell Signaling Technology Inc)

Journal: EXCLI Journal

Article Title: Long-term Western diet feeding impairs hepatic vitamin D metabolism and promotes bone loss in mice

doi: 10.17179/excli2025-9085

Figure Lengend Snippet: Histological assessment of bone composition. (A) Representative image of perilipin-positive adipocytes (arrowheads) from SD and WD mice at week 48. Scale bars: 20 µm. The average nuclear area was 14.67 µm² in SD mice and 14.22 µm² in WD mice. (B) Quantitative analysis of perilipin-positive adipocytes from the two groups. (C) Representative image of CD163-positive macrophage (arrowheads) from SD and WD mice at week 48. (D) Quantitative analysis of CD163-positive macrophages from the two groups. Scale bars: 20 µm. The average nuclear area was 14.43 µm² in SD mice and 14.50 µm² in WD mice. Data are shown as box-and-whisker plots. Statistical analysis was performed using the Mann-Whitney U test. * p < 0.05, ** p < 0.01 as compared to SD group. SD = standard diet, WD = Western diet.

Article Snippet: For immunohistochemical staining, sections were incubated for 1 h with a monoclonal rabbit anti-mouse antibody against perilipin (1:200; Cell Signaling Technology, Danvers, MA, USA) and a rabbit anti-CD163 (1:300; Abcam, Cambridge, UK) as the primary antibody.

Techniques: Whisker Assay, MANN-WHITNEY, Western Blot

Journal: Cell Reports Medicine

Article Title: α2δ1-mediated maladaptive sensory plasticity disrupts adipose tissue homeostasis following spinal cord injury

doi: 10.1016/j.xcrm.2024.101525

Figure Lengend Snippet:

Article Snippet: Rabbit Perilipin-1 (D1D8) XP® mAb, 1:2000 (IHC) , Cell Signaling Technology , Cat #9349; RRID: AB_10829911.

Techniques: Flow Cytometry, Recombinant, Plasmid Preparation, Saline, Protease Inhibitor, Staining, Enzyme-linked Immunosorbent Assay, H&E Stain, Bicinchoninic Acid Protein Assay, cDNA Synthesis, SYBR Green Assay, Software, Imaging, Microscopy, Real-time Polymerase Chain Reaction, Blocking Assay, Membrane

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Unloading-Induced Skeletal Interoception Alters Hypothalamic Signaling to Promote Bone Loss and Fat Metabolism.

doi: 10.1002/advs.202305042

Figure Lengend Snippet: Figure 3. Bone loss and lipolysis activity in HU mice could be attenuated by a non-selective 𝛽-blocker, propranolol (Prop). Control and HU mice were injected with saline or propranolol at a dose of 0.5 mg kg−1 per day for 2 weeks. a–c) Representative μCT images (a) and quantitative analysis of trabecular BV/TV (b) and Trabecular number (Tb.N) (c) in different treatment groups. Scale bar = 0.5 mm. N = 5. d) Representative immunofluorescent staining of osteocalcin (OCN)-positive cells (red) and e) their quantitative analysis in femoral bone marrow area of different treatment groups. Scale bar = 40 μm. N = 5. f) Representative immunofluorescent staining of perilipin-positive cells (green) and (g) their quantitative analysis in femoral bone marrow area of different treatment groups. Scale bar = 40 μm. N = 5. h) Representative TRAP staining images and i) quantification of TRAP+ cells in femoral bone marrow area of different treatment groups. Scale bar = 40 μm. N = 5. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with the Dunnet post hoc test.

Article Snippet: Briefly, the sections were incubated with primary antibodies to rabbit Tyrosine hydroxylase (Millipore Sigma, ab152, 1:100), goat NPY (Novus, NBP1-46535, 1:100), rabbit Cathepsin K (Abcam, ab19027, 1:100), rabbit Osteocalcin (Origene, BP710, 1:100), rabbit pAMPK (Cell Signaling Technology, 2535s, 1:100), rabbit Perilipin (Cell Signaling Technology, D1D8, 1:100), goat Perilipin (Abcam, ab60269, 1:100), rabbit Osterix (Abcam, ab22552, 1:100) at 4 °C overnight.

Techniques: Activity Assay, Control, Injection, Saline, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Unloading-Induced Skeletal Interoception Alters Hypothalamic Signaling to Promote Bone Loss and Fat Metabolism.

doi: 10.1002/advs.202305042

Figure Lengend Snippet: Figure 5. Depletion of TH-positive sympathetic nerves or inhibition of NE release blunts bone resorption in HU mice. a,b) ELISA analysis of nore- pinephrine (NE) level in bone marrow (a) and cortical bone (b) of mice with different treatments. N = 5. c) Representative immunofluorescent staining of TH-positive nerve fibers (red) from femoral bone marrow and d) their quantitative analysis in different treatment groups. e–h) Representative μCT images (e) and quantitative analysis of different bone parameters, including Po.tot (f), TMD (g) of cortical bone, and BV/TV (h) of trabecular bone in different treatment groups. Scale bar = 0.5 mm. N = 5. i) Representative immunofluorescent staining of perilipin-positive cells (green) and j) their quan- titative analysis in femoral bone marrow area of different treatment groups. Scale bar = 40 μm. N = 5. k) Representative immunofluorescent staining of OCN-positive cells (red) and l) their quantitative analysis in femoral bone marrow area of different treatment groups. Scale bar = 40 μm. N = 5. m) Representative TRAP staining (purple) images at femoral mid-shaft of cortical bone and n) quantification of TRAP+ osteocytes on femoral cortical bone in different treatment groups. Scale bar = 40 μm. N = 5. o) Representative immunofluorescent staining of Ctsk (green) at femoral mid-shaft of cortical bone and p) quantification of Ctsk+ osteocytes on femoral cortical bone in different treatment groups. Scale bar = 40 μm. N = 5. q) Representative Alcian blue staining and r) quantification of osteocyte lacuna area of cortical bone in different treatment groups. Scale bar = 40 μm. N = 5. The conjugates of F127/Vehicle made by 30% F127 (30% w/v F127 in PBS); the conjugates of F127/6-OHDA made by 6-OHDA in 30% F127, the dose of 6-OHDA was 10 mg kg−1 per day; the conjugates of F127/guanethidine made by guanethidine in 30% F127, the dose of guanethidine was 10 mg kg−1 per day. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with the Dunnet post hoc test.

Article Snippet: Briefly, the sections were incubated with primary antibodies to rabbit Tyrosine hydroxylase (Millipore Sigma, ab152, 1:100), goat NPY (Novus, NBP1-46535, 1:100), rabbit Cathepsin K (Abcam, ab19027, 1:100), rabbit Osteocalcin (Origene, BP710, 1:100), rabbit pAMPK (Cell Signaling Technology, 2535s, 1:100), rabbit Perilipin (Cell Signaling Technology, D1D8, 1:100), goat Perilipin (Abcam, ab60269, 1:100), rabbit Osterix (Abcam, ab22552, 1:100) at 4 °C overnight.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Unloading-Induced Skeletal Interoception Alters Hypothalamic Signaling to Promote Bone Loss and Fat Metabolism.

doi: 10.1002/advs.202305042

Figure Lengend Snippet: Figure 6. HU mice display greater NPY expression and inhibition of NPY Y1R blunts the negative effects on bone and fat metabolism of unloading. a) ELISA analysis of NPY level in serum of control and HU mice. N = 6. b) Quantitative analysis of food intake for control and HU mice. N = 6. c) Quantitative analysis of body weight for control and HU mice. N = 6. d–f) Quantitative analysis of the weight of the soleus (d), the gonadal (e), and inguinal fat pads (f) isolated from control and HU mice. N = 6. g) ELISA analysis of free fatty acid level in serum from control and HU mice. N = 6. h) Representative immunofluorescent staining of perilipin-positive cells (green) and their quantitative analysis i) in femoral bone marrow area of control and HU mice. Scale bar = 40 μm. N = 6. j,k) mRNA expression of Lipe (j) and Pnpla2 (k) by RT-qPCR in white adipose tissues of control and HU mice. N = 6. l,m) Representative co-immunofluorescent staining of pAMPK (red) and perilipin (green) from gonadal white adipose tissues (l) and quantitative analysis of pAMPK+/Perilipin+ adipocytes per area (m) in control and HU mice. Scale bar = 40 μm. N = 6. n) Quantitative analysis of food intake for HU mice treated with vehicle and BIBO3304 (1 mg kg−1 per day) every day for 2 weeks. N = 6. o) Quantitative analysis of body weight for HU mice treated with vehicle and BIBO3304. N = 6. p–r) Quantitative analysis of the weight of the soleus (p) and gonadal fat pads (q), and the inguinal fat pads (r) isolated from HU mice treated with vehicle and BIBO3304. N = 6. s,t) mRNA expression of Lipe (s) and Pnpla2 (t) by RT-qPCR in white adipose tissues of HU mice treated with vehicle and BIBO3304. N = 6. u,v) Representative μCT-detected OsO4-stained images of decalcified femurs (u) and quantitative analysis of the number of adipocytes (Ad.N) (v) in HU mice treated with vehicle and BIBO3304. N = 6. Scale bar = 0.5 mm. w–y) Representative μCT images (w) and quantitative analysis of different bone parameters, including trabecular number (Tb.N) (x) and trabecular BV/TV (y) in HU mice treated with vehicle and BIBO3304, respectively. Scale bar = 1 mm. N = 6. z,z1) Representative immunofluorescent staining of OCN (red) (z) and quantification of OCN-positive cells (z1) in femoral bone marrow area of HU mice treated with vehicle and BIBO3304, respectively. N = 6. Scale bar = 40 μm. Data are presented as mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t-test.

Article Snippet: Briefly, the sections were incubated with primary antibodies to rabbit Tyrosine hydroxylase (Millipore Sigma, ab152, 1:100), goat NPY (Novus, NBP1-46535, 1:100), rabbit Cathepsin K (Abcam, ab19027, 1:100), rabbit Osteocalcin (Origene, BP710, 1:100), rabbit pAMPK (Cell Signaling Technology, 2535s, 1:100), rabbit Perilipin (Cell Signaling Technology, D1D8, 1:100), goat Perilipin (Abcam, ab60269, 1:100), rabbit Osterix (Abcam, ab22552, 1:100) at 4 °C overnight.

Techniques: Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Control, Isolation, Staining, Quantitative RT-PCR, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Unloading-Induced Skeletal Interoception Alters Hypothalamic Signaling to Promote Bone Loss and Fat Metabolism.

doi: 10.1002/advs.202305042

Figure Lengend Snippet: Figure 7. Knockdown of NPY mRNA expression in the ARC leads to alterations in energy expenditure with limited effects on TH expression and bone resorption. a) Representative immunofluorescent staining of NPY (green) in ARC of the hypothalamus and b) its quantitative analysis in HU mice treated with AAV-Control and AAV-shNPY, respectively. Higher magnifications of selected areas are shown at the lower side of corresponding panels. N = 6. Scale bar = 40 μm. c) mRNA expression of Npy by RT-qPCR in the hypothalamus of HU mice treated with AAV-Control and AAV-shNPY. N = 6. d) ELISA analysis of NPY level in serum of HU mice treated with AAV-Control and AAV-shNPY. N = 6. e) Representative immunofluorescent staining of TH-positive neurons and nerve fibers (red) from PVN of hypothalamus and f) their quantitative analysis in HU mice treated with AAV-Control and AAV- shNPY. Scale bar = 40 μm. N = 6. g) ELISA analysis of NE level in serum of HU mice treated with AAV-Control and AAV-shNPY. N = 6. h) Quantitative analysis of food intake for HU mice treated with AAV-Control and AAV-shNPY, respectively. N = 6. i) Quantitative analysis of body weight for HU mice treated with AAV-Control and AAV-shNPY, respectively. N = 6. j–l) Quantitative analysis of the weight of the soleus (j), the gonadal (k), and inguinal fat pads (l) isolated from HU mice treated with AAV-Control and AAV-shNPY, respectively. N = 6. m) ELISA analysis of free fatty acid level in serum from HU mice treated with AAV-Control and AAV-shNPY. N = 6. n,o) mRNA expression of Lipe (n) and Pnpla2 (o) by RT-qPCR in white adipose tissues of HU mice treated with AAV-Control and AAV-shNPY. N = 6. p) Representative co-immunofluorescent staining and (q) quantitative analysis of pAMPK (red) and perilipin (green) from gonadal white adipose tissues of HU mice treated with AAV-Control and AAV-shNPY. Scale bar = 40 μm. N = 6. r) Representative μCT-detected OsO4-stained images of decalcified femurs and s)quantitative analysis of Ad.N in HU mice treated with AAV-Control and AAV-shNPY. Scale bar = 1 mm. N = 6. t–x) Representative μCT images (t) and quantitative analysis of different bone parameters, including Po.tot (u) and Cor.Th (v) of cortical bone, BV/TV (w), and Tb.N (x) of trabecular bone in HU mice treated with AAV-Control and AAV-shNPY. Scale bar = 0.5 mm. N = 6. y) Representative immunofluorescent staining of OCN (red) and (z) quantification of perilipin-positive and OCN-positive cells in femoral bone marrow area of HU mice treated with AAV-Control and AAV-shNPY. Scale bar = 40 μm. N = 6. z1) Diagram showing that decreased skeletal PGE2/EP4 ascending signal increased the expression of both sympathetic tone and NPY level in the hypothalamus as the independent descending interoceptive signal for bone and fat metabolism in the HU model. Data are presented as mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t-test.

Article Snippet: Briefly, the sections were incubated with primary antibodies to rabbit Tyrosine hydroxylase (Millipore Sigma, ab152, 1:100), goat NPY (Novus, NBP1-46535, 1:100), rabbit Cathepsin K (Abcam, ab19027, 1:100), rabbit Osteocalcin (Origene, BP710, 1:100), rabbit pAMPK (Cell Signaling Technology, 2535s, 1:100), rabbit Perilipin (Cell Signaling Technology, D1D8, 1:100), goat Perilipin (Abcam, ab60269, 1:100), rabbit Osterix (Abcam, ab22552, 1:100) at 4 °C overnight.

Techniques: Knockdown, Expressing, Staining, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Two Tailed Test