Journal: bioRxiv

Article Title: Quantitative Multicolored Deep Imaging of Human Bones Reveals a Composite Osteo-Sinusoidal Niche for Mesenchymal Stromal Cells

doi: 10.1101/2025.10.07.680053

Figure Lengend Snippet: a,b , Colocalization of LEPR protein (antibody staining, pseudocoloured yellow) and CXCL12 mRNA (HCR RNA-FISH, red) in representative 2D optical sections from ( a ) a 3D light-sheet scan and ( b ) a confocal scan of tissue-cleared human bone. Left panels show LEPR omitted; right panels include all channels. Nuclei (YOPRO, green) and bone matrix (autofluorescence at 405 nm, cyan) were visualized in ( a ) but not ( b ) due to sample bleaching. c , Confocal image of a 6-μm thick section from decalcified human bone stained for LEPR (green), CXCL12 mRNA (red), and counterstained with DAPI (blue). Single and merged channels are shown. d–f , Nuclear staining of aged human bone with YOPRO1 (green). d, Raw YOPRO+ signals in a light-sheet scan. e, YOPRO+ nuclei overlaid with cell positions detected by the Imaris Spot function. f, Optical sections showing overlay of YOPRO+ nuclei with segmented cell masks (grey circles). g–l , Detection of nucleated CXCL12+ cells in aged bone. g–i, Representative 2D optical sections from a 3D light-sheet scan showing CXCL12 mRNA (HCR, red), CD31+ vessels (yellow), and YOPRO1+ nuclei (green). CXCL12 signal is omitted in g and present in h ; i , colocalized CXCL12+YOPRO+ cells detected by Imaris Spot function are marked by grey circles. j–l , 3D projections of the same region as a raw scan ( j ) and detected double-positive cells at low ( k ) and high ( l ) magnification, with double-positive cells represented as green spheres. j , CD31 and YOPRO channels only; k,l , all channels including CXCL12. All images are cropped from large tissue volumes (∼2 × 2 × 2 mm) scanned by light-sheet microscopy.

Article Snippet: Following HCR, tissue sections were blocked using the same staining buffer as used in DeepBone, then incubated with primary antibodies against LEPR (conjugated to AlexaFluor488; #MAB867, R&D Systems) or Ki67 (conjugated to AlexaFluor546; SolA15, Thermofisher).

Techniques: Staining, Microscopy

Journal: Endocrine

Article Title: Leptin at gender-specific concentrations does not affect glucose transport, expression of glucose transporters and leptin receptors in human lymphocytes

doi: 10.1007/s12020-014-0435-3

Figure Lengend Snippet: Immunocytochemical detection of GLUT4 and leptin receptors in lymphocytes. a Negative control for females lymphocytes incubated without the first antibody. The arrow shows an exemplary negative cell. b Females lymphocytes incubated in elevated leptin concentration showed positive reaction against GLUT4. The arrow shows an exemplary lymphocyte with positive reaction against GLUT4. c Negative control for males lymphocytes incubated without the first antibody. The arrow shows an exemplary negative cell. d Males lymphocytes incubated in elevated leptin concentration showed positive reaction against leptin receptor. The arrow shows an lymphocyte with positive reaction against leptin receptor. The arrow with a star shows an lymphocyte without positive reaction against leptin receptor

Article Snippet: After 30 min mouse monoclonal antibody (1:200) against extracellular domain of human leptin receptor (R&D Systems) was added and then horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000) (Chemicon International Inc. Ca).

Techniques: Negative Control, Incubation, Concentration Assay

Journal: Endocrine

Article Title: Leptin at gender-specific concentrations does not affect glucose transport, expression of glucose transporters and leptin receptors in human lymphocytes

doi: 10.1007/s12020-014-0435-3

Figure Lengend Snippet: Percent of females and males lymphocytes showing expression of GLUT proteins and leptin receptors. The results ( n = 20 for each gender) were presented as mean ± SD. Statistical analysis was performed by two-way ANOVA with replication. Differences between control and leptin-treated lymphocytes were not statistically significant Normal leptin refers to physiologic level: 4 ng/ml for men and 8 ng/ml for women. Elevated leptin refers to low pathophysiologic level: 8 ng/ml for men and 32 ng/ml for women

Article Snippet: After 30 min mouse monoclonal antibody (1:200) against extracellular domain of human leptin receptor (R&D Systems) was added and then horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000) (Chemicon International Inc. Ca).

Techniques: Expressing, Control

Journal: Endocrine

Article Title: Leptin at gender-specific concentrations does not affect glucose transport, expression of glucose transporters and leptin receptors in human lymphocytes

doi: 10.1007/s12020-014-0435-3

Figure Lengend Snippet: Mean intensity of fluorescence of particular GLUT proteins and leptin receptor (LR) in females and males lymphocytes. The results ( n = 20 for each gender) were presented as mean ± SD. Statistical analysis was performed by two-way ANOVA with replication. Differences between control and leptin-treated lymphocytes were not statistically significant. Normal leptin refers to physiologic level: 4 ng/ml for men and 8 ng/ml for women. Elevated leptin refers to low pathophysiologic level: 8 ng/ml for men and 32 ng/ml for women

Article Snippet: After 30 min mouse monoclonal antibody (1:200) against extracellular domain of human leptin receptor (R&D Systems) was added and then horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000) (Chemicon International Inc. Ca).

Techniques: Fluorescence, Control

Journal: Endocrine

Article Title: Leptin at gender-specific concentrations does not affect glucose transport, expression of glucose transporters and leptin receptors in human lymphocytes

doi: 10.1007/s12020-014-0435-3

Figure Lengend Snippet: Representative histograms of GLUT1 from flow cytometry analysis of females samples incubated in different leptin concentration. The first antibody used was polyclonal rabbit antibody against GLUT1. The secondary antibody was swine anti-rabbit IgG-FITC. a control without leptin; b normal leptin concentration; c elevated leptin concentration; d control sample (negative) without antibody against GLUT1

Article Snippet: After 30 min mouse monoclonal antibody (1:200) against extracellular domain of human leptin receptor (R&D Systems) was added and then horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000) (Chemicon International Inc. Ca).

Techniques: Flow Cytometry, Incubation, Concentration Assay, Control

Journal:

Article Title: Unique leptin trafficking by a tailless receptor

doi: 10.1096/fj.09-143487

Figure Lengend Snippet: Scenario for construction and characterization of mutant leptin receptors. A) Schematic drawing of the deletion constructs directed to different functional domains of ObRa and ObRb. Both receptor isoforms share the signal peptide (aa 1–21), an extracellular domain (aa 22–839), the single transmembrane domain (aa 840–860), and the first 29 aa of the cytoplasmic tail until aa 889. Deletion constructs with the last aa labeled in the protein sequence are shown. B) Truncated ObR constructs were generated by PCR cloning and cloned into a pcDNA3.1(−) vector at XbaI and HindIII restriction enzyme sites. Correct sizes of insertions were verified by restriction enzyme digestion following agarose gel electrophoresis. C) Truncated ObR constructs were expressed in HEK293 cells. Protein lysates 24 h after transient transfection showed efficient protein expression at the predicted molecular weight. Last lane is a negative control transfected with pcDNA3.2 vector. D) Immunocytochemistry of ObR in HEK293 cells transfected by ObR860 (tailless receptor) and ObRa. Both constructs showed cell surface expression of ObR.

Article Snippet: To test the level of ObR protein expression, 80 μg of protein extracted from ObR-transfected HEK293 cells was loaded onto 7% polyacrylamide gels and electrophoresed for 2 h. The proteins were transferred to nitrocellulose membrane (Bio-Rad Laboratories), and probed with a primary goat antibody against the shared extracellular domain of mouse leptin receptor (R&D Systems).

Techniques: Mutagenesis, Construct, Functional Assay, Labeling, Sequencing, Generated, PCR Cloning, Clone Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Transfection, Expressing, Molecular Weight, Negative Control, Immunocytochemistry

Journal:

Article Title: Unique leptin trafficking by a tailless receptor

doi: 10.1096/fj.09-143487

Figure Lengend Snippet: Test of intracellular leptin signaling mediated by mutant leptin receptors. A) Among all ObRs, only ObRb mediated STAT3 activation 15 min after leptin treatment. Housekeeping gene β-actin was probed as a loading control. B) Among ObR constructs, ObR1137 induced a 268-fold increase of STAT1 transcriptional activation, shown by luciferase reporter activity. This contrasts with less activation by ObR860, ObR889, ObR895, and ObRc, and a 2439-fold increase mediated by ObRb.

Article Snippet: To test the level of ObR protein expression, 80 μg of protein extracted from ObR-transfected HEK293 cells was loaded onto 7% polyacrylamide gels and electrophoresed for 2 h. The proteins were transferred to nitrocellulose membrane (Bio-Rad Laboratories), and probed with a primary goat antibody against the shared extracellular domain of mouse leptin receptor (R&D Systems).

Techniques: Mutagenesis, Activation Assay, Control, Construct, Luciferase, Activity Assay

Journal:

Article Title: Unique leptin trafficking by a tailless receptor

doi: 10.1096/fj.09-143487

Figure Lengend Snippet: Comparison of ObR860 and ObRa in mediating leptin trafficking in HEK293 cells. A) Most ObR860 immunoreactivity (green) was located at cell surface after leptin stimulation for 2 min, at which time Alexa555-leptin (red) showed complete colocalization with the receptor. At 15 min, Alexa555-leptin remained colocalized with ObR860, but mainly intracellularly. B) Immunoreactivity of ObRa was present both at cell surface and in cytosplasmic vesicles 2 min after leptin treatment. At this time, Alexa555-leptin (red) showed complete colocalization with ObRa (green). Fifteen minutes later, most of the Alexa555-leptin was colocalized with ObRa in intracellular vesicles.

Article Snippet: To test the level of ObR protein expression, 80 μg of protein extracted from ObR-transfected HEK293 cells was loaded onto 7% polyacrylamide gels and electrophoresed for 2 h. The proteins were transferred to nitrocellulose membrane (Bio-Rad Laboratories), and probed with a primary goat antibody against the shared extracellular domain of mouse leptin receptor (R&D Systems).

Techniques: Comparison

Journal:

Article Title: Unique leptin trafficking by a tailless receptor

doi: 10.1096/fj.09-143487

Figure Lengend Snippet: Test of membrane microdomains involved in leptin-induced internalization of the ligand-receptor complex in HEK293 cells. A) Effects of Dynasore and chlorpromazine on 125I-leptin endocytosis. Dynasore inhibited leptin endocytosis in both ObRa- and ObR860-overexpressing cells in a dose-dependent manner. *P < 0.05; ***P < 0.005. B) Cellular distribution of Alexa555-leptin was similar in HEK293 cells overexpressing either ObRa or ObR860. In DMSO vehicle-treated cells, 20 min after initiation of endocytosis, leptin fluorescence was seen at both cell surface and vesicular compartments in cytoplasm. High dose of Dynasore (160 μM) blocked internalization in both ObRa- and ObR860-overexpressing cells. Chlorpromazine also blocked internalization of leptin mediated by ObR860 but not by ObRa. Lower dose of Dynasore (80 μM), nystatin, or filipin did not alter trafficking pattern of leptin.

Article Snippet: To test the level of ObR protein expression, 80 μg of protein extracted from ObR-transfected HEK293 cells was loaded onto 7% polyacrylamide gels and electrophoresed for 2 h. The proteins were transferred to nitrocellulose membrane (Bio-Rad Laboratories), and probed with a primary goat antibody against the shared extracellular domain of mouse leptin receptor (R&D Systems).

Techniques: Membrane, Fluorescence

Journal: Central European Journal of Immunology

Article Title: Experimental immunology Differences in the expression of leptin receptors on bone marrow and peripheral blood cells

doi: 10.5114/ceji.2012.32721

Figure Lengend Snippet: Fig. 1. Electronically differentiation according forward scat- ter (FS) vs. side scatter (SS) population of bone marrow cells (A) and peripheral blood leukocytes (B). Percentage of mononuclear cells (lymphocytes – gate A and monocytes – gate B) and granulocytes (gate C) are given in cytograms. Cytogram C shows granulocytes isolated from peripheral blood and stained with anti-CD13/CD33 (FL2) and mouse anti OB-R monoclonal antibodies. Goat antibody against mouse IgG con- jugated with fluoresceine (FL1) was used as the secondary anti- body. Area U1 contained CD13/33+Ob-R-cells; area U2 con- tained double positive cells: CD13/33+OB-R+ and in U3 area are cells double negative. In U4 area are another cells presented after isolation that are Ob-R+

Article Snippet: OB-R receptor was stained according to indirect immunofluorescence method using murine monoclonal IgG2A antibody against human leptin receptor manufactured by R&D Systems and secondary FITC-conjugated AffiniPure Goat Anti-Mouse IgG manufactured by Jackson ImmunoResearch Laboratories, Inc. Monocytic cells lineage was identified using anti CD4 and CD14 antibodies, while granulocytic lineage was identified with anti CD13 and CD33 antibodies.

Techniques: Isolation, Staining, Bioprocessing

Journal: Current Zoology

Article Title: Leptin/leptin receptor system in the regulation of reproductive functions and stress response in the European beaver

doi: 10.1093/cz/zoy030

Figure Lengend Snippet: The sequences of primers used for identification of leptin receptor (Ob-R) cDNA in PCR (A) or leptin receptor and reference genes: β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in quantitative real-time PCR (B)

Article Snippet: The sections were incubated with primary rabbit polyclonal antibodies against leptin receptor (1:50, Abcam ® UK) and with secondary anti-mouse/rabbit antibodies (ImmPRESS Universal Reagent Anti-Mouse/Rabbit Ig, Vector Laboratories, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Current Zoology

Article Title: Leptin/leptin receptor system in the regulation of reproductive functions and stress response in the European beaver

doi: 10.1093/cz/zoy030

Figure Lengend Snippet: The sequence of leptin receptor in beavers and its homology with the European rabbit (XM_008265114.1), rat (NM_012596.1), mouse (NM_146146.2), pig (NM_001024587.1), and with human (NM_001198689.1) Ob-R sequences.

Article Snippet: The sections were incubated with primary rabbit polyclonal antibodies against leptin receptor (1:50, Abcam ® UK) and with secondary anti-mouse/rabbit antibodies (ImmPRESS Universal Reagent Anti-Mouse/Rabbit Ig, Vector Laboratories, USA).

Techniques: Sequencing

Journal: Current Zoology

Article Title: Leptin/leptin receptor system in the regulation of reproductive functions and stress response in the European beaver

doi: 10.1093/cz/zoy030

Figure Lengend Snippet: The expression of leptin receptor gene in the medio-basal hypothalamus (MBH; A , B ), pituitary gland ( C , D ), gonads ( E , F ), uterus ( G , H ), adrenal cortex ( I , J ), and white adipose tissue (WAT; K , L ) in males of the European beaver during different seasons of the reproductive activity (April—breeding, July—post-breeding, and November—pre-breeding). Different letters indicate significant differences ( P < 0.05) between the columns representing various groups of animals, whereas the same letters indicate a lack of differences between columns.

Article Snippet: The sections were incubated with primary rabbit polyclonal antibodies against leptin receptor (1:50, Abcam ® UK) and with secondary anti-mouse/rabbit antibodies (ImmPRESS Universal Reagent Anti-Mouse/Rabbit Ig, Vector Laboratories, USA).

Techniques: Expressing, Activity Assay

Journal: Current Zoology

Article Title: Leptin/leptin receptor system in the regulation of reproductive functions and stress response in the European beaver

doi: 10.1093/cz/zoy030

Figure Lengend Snippet: The leptin receptor protein localization in the representative tissue sections of MBH ( A ), pituitary ( B ), gonads ( C , D ), uterus ( E ), and adrenal gland ( F ). The leptin receptor protein is marked in brown color (DAB). Hematoxylin (violet color) was applied for nuclei staining. The pictures (a1–f1) indicate negative controls (the procedure without the primary antibody). The pictures (A2–F2) indicate leptin receptor protein localization in the tested tissues of female and male (D2) captured in April (breeding). The pictures (A3–F3) indicate leptin receptor protein localization in the tested tissues of female and male (d3) captured in July (post-breeding). The pictures (A4–F4) indicate leptin receptor protein localization in the tested tissues of female and male (d4) captured in November (pre-breeding).

Article Snippet: The sections were incubated with primary rabbit polyclonal antibodies against leptin receptor (1:50, Abcam ® UK) and with secondary anti-mouse/rabbit antibodies (ImmPRESS Universal Reagent Anti-Mouse/Rabbit Ig, Vector Laboratories, USA).

Techniques: Staining

Journal: Scientific Reports

Article Title: CB2 receptor deletion on myeloid cells enhanced mechanical allodynia in a mouse model of neuropathic pain

doi: 10.1038/s41598-019-43858-4

Figure Lengend Snippet: Coexpression of leptin receptor (red) with F4/80 (green). Sciatic nerve tissue of WT, CB2-LysM and CB2-Syn mice was stained 14 days post SNL. Leptin receptor signal was highest and co-labeled with the macrophage marker F4/80 in ipsilateral sciatic nerve of CB2-LysM mice compared to WT or CB2-Syn mice. Arrows show co-labeled cells. (scale = 250 µm, 63x zoom scale = 50 µm).

Article Snippet: Secondary antibodies used were against leptin receptor antibody (705-166-147, Jackson Immuno Research Laboratories, 1:250) and F4/80 antibody (A21208, Life technologies, 1:250).

Techniques: Staining, Labeling, Marker