primary anti lcn2 antibody  (R&D Systems)

Journal: Frontiers in Endocrinology

Article Title: Ovaries of estrogen receptor 1-deficient mice show iron overload and signs of aging

doi: 10.3389/fendo.2024.1325386

Figure Lengend Snippet: Hemosiderin deposits in ovarian tissues. Formalin-fixated paraffin-embedded ovarian tissues section of wild type (WT, n=8) and Esr1 -deficient (n=8) animals were used for staining. Serial tissue slices of WT (A) and Esr1 -deficient (B) ovaries were stained with Hematoxylin-Eosin (HE) or Perls Prussian Blue (PPB) to detect iron in form of hemosiderin. Hemosiderin can be seen in HE staining as brown-gold deposits and turns blue with PPB. The scale bars equal to 250 µm (solid boarder) or 50 µm (dashed border). Hemosiderin accumulations were found in ovarian stroma of Esr1 -deficient animals but not in WTs and were localized around hemorrhagic cysts (C) . The scale bars equal to 100 µm. (D) Immunohistochemical localization of lipocalin 2 (LCN2) was combined with PPB staining to examine whether iron-positive cells co-localize with LCN2-positive cells. WT (n=3) and Esr1 -deficient (n=4) ovaries were stained, showing no co-localization of iron and LCN2. Normal goat IgG was used as a negative control instead of the primary antibody. The scale bars equal to 500 µm (solid boarder) or 50 µm (dotted boarder and dashed border).

Article Snippet: To block non-specific antibody binding sites, tissue sections were incubated in 5% normal rabbit serum (#X0902, Agilent Technologies) in blocking solution (1% BSA, 0.1% cold fish gelatin, 0.1% Triton-X-100, 0.05% Tween ® 20 in PBS) for 90 min. Primary anti-LCN2 Antibody (#AF3508, R&D Systems, Minneapolis, MN, USA) was diluted 1:40 in blocking solution and incubated on slides at 4°C overnight.

Techniques: Staining, Immunohistochemical staining, Negative Control

Journal: Frontiers in Endocrinology

Article Title: Ovaries of estrogen receptor 1-deficient mice show iron overload and signs of aging

doi: 10.3389/fendo.2024.1325386

Figure Lengend Snippet: Macrophages in ovarian tissues. Wild type (WT) and Esr1 -deficient ovarian tissues were either used for mRNA (WT, n=7; Esr1 –/– , n=5-6), protein analysis (WT, n=3; Esr1 –/– , n=3) or immunofluorescence staining (WT, n=3; Esr1 –/– , n=3). (A) Relative mRNA expression of pan-macrophage markers Cd68 and Adgre were detected by RT-qPCR. (B) Western blot analysis was used to detect CD68 protein expression, which was quantified densitometrically and plotted relative to β-actin expression. F4/80 (green) and LCN2 (red) protein expression was visualized by immunofluorescence staining with nuclear DAPI counterstaining in (C) WT and Esr1 -deficient (D) ovaries. (E) Normal rat and goat IgG were used as a negative control instead of the primary antibodies. The scale bars equal to 100 µm (solid boarder) or 50 µm (dashed border). (F) Relative mRNA expression of Nos2 and Il1r1 , as well as Arg1 , Cd163 and Mrc (G) were determined by RT-qPCR. All data (A-B, F-G) are displayed as mean ± SD. For statistical analysis, Students t -test was done. Significant differences between groups are marked with asterisks: * p <0.05, ** p <0.01, *** p <0.001, ns =not significant.

Article Snippet: To block non-specific antibody binding sites, tissue sections were incubated in 5% normal rabbit serum (#X0902, Agilent Technologies) in blocking solution (1% BSA, 0.1% cold fish gelatin, 0.1% Triton-X-100, 0.05% Tween ® 20 in PBS) for 90 min. Primary anti-LCN2 Antibody (#AF3508, R&D Systems, Minneapolis, MN, USA) was diluted 1:40 in blocking solution and incubated on slides at 4°C overnight.

Techniques: Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Frontiers in Endocrinology

Article Title: Ovaries of estrogen receptor 1-deficient mice show iron overload and signs of aging

doi: 10.3389/fendo.2024.1325386

Figure Lengend Snippet:

Article Snippet: To block non-specific antibody binding sites, tissue sections were incubated in 5% normal rabbit serum (#X0902, Agilent Technologies) in blocking solution (1% BSA, 0.1% cold fish gelatin, 0.1% Triton-X-100, 0.05% Tween ® 20 in PBS) for 90 min. Primary anti-LCN2 Antibody (#AF3508, R&D Systems, Minneapolis, MN, USA) was diluted 1:40 in blocking solution and incubated on slides at 4°C overnight.

Techniques: Clinical Proteomics, Mass Spectrometry, Saline, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Augmented Lipocalin-2 Is Associated with Chronic Obstructive Pulmonary Disease and Counteracts Lung Adenocarcinoma Development

doi: 10.1164/rccm.202004-1079oc

Figure Lengend Snippet: Figure 1. Increased Lcn2 during LUAD development in Gprc5a-/- mice.

Article Snippet: Following antigen retrieval at 100°C for 20 minutes (Bond ER Solution #1, Leica Biosystems), four-micron tissue sections were incubated with anti-LCN2 polyclonal goat primary antibody (AF1757, 1:1000 dilution, R&D Systems, Minneapolis, MN) and detected using the Bond Polymer Refine Detection kit (DS9800, Leica Biosystems).

Techniques:

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Augmented Lipocalin-2 Is Associated with Chronic Obstructive Pulmonary Disease and Counteracts Lung Adenocarcinoma Development

doi: 10.1164/rccm.202004-1079oc

Figure Lengend Snippet: Figure 2. Elevated LCN2 expression in human LUAD.

Article Snippet: Following antigen retrieval at 100°C for 20 minutes (Bond ER Solution #1, Leica Biosystems), four-micron tissue sections were incubated with anti-LCN2 polyclonal goat primary antibody (AF1757, 1:1000 dilution, R&D Systems, Minneapolis, MN) and detected using the Bond Polymer Refine Detection kit (DS9800, Leica Biosystems).

Techniques: Expressing

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Augmented Lipocalin-2 Is Associated with Chronic Obstructive Pulmonary Disease and Counteracts Lung Adenocarcinoma Development

doi: 10.1164/rccm.202004-1079oc

Figure Lengend Snippet: Figure 3. Upregulation of LCN2 in human COPD and COPD-associated LUAD.

Article Snippet: Following antigen retrieval at 100°C for 20 minutes (Bond ER Solution #1, Leica Biosystems), four-micron tissue sections were incubated with anti-LCN2 polyclonal goat primary antibody (AF1757, 1:1000 dilution, R&D Systems, Minneapolis, MN) and detected using the Bond Polymer Refine Detection kit (DS9800, Leica Biosystems).

Techniques:

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Augmented Lipocalin-2 Is Associated with Chronic Obstructive Pulmonary Disease and Counteracts Lung Adenocarcinoma Development

doi: 10.1164/rccm.202004-1079oc

Figure Lengend Snippet: Figure 4. Loss of Lcn2 augments LUAD development in tobacco carcinogen-exposed Gprc5a-/- mice.

Article Snippet: Following antigen retrieval at 100°C for 20 minutes (Bond ER Solution #1, Leica Biosystems), four-micron tissue sections were incubated with anti-LCN2 polyclonal goat primary antibody (AF1757, 1:1000 dilution, R&D Systems, Minneapolis, MN) and detected using the Bond Polymer Refine Detection kit (DS9800, Leica Biosystems).

Techniques:

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Augmented Lipocalin-2 Is Associated with Chronic Obstructive Pulmonary Disease and Counteracts Lung Adenocarcinoma Development

doi: 10.1164/rccm.202004-1079oc

Figure Lengend Snippet: Figure 5. Loss of Lcn2 in tobacco carcinogen-exposed Gprc5a-/- mice promotes gene expression programs associated with reduced anti-tumor immunity.

Article Snippet: Following antigen retrieval at 100°C for 20 minutes (Bond ER Solution #1, Leica Biosystems), four-micron tissue sections were incubated with anti-LCN2 polyclonal goat primary antibody (AF1757, 1:1000 dilution, R&D Systems, Minneapolis, MN) and detected using the Bond Polymer Refine Detection kit (DS9800, Leica Biosystems).

Techniques: Gene Expression

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Augmented Lipocalin-2 Is Associated with Chronic Obstructive Pulmonary Disease and Counteracts Lung Adenocarcinoma Development

doi: 10.1164/rccm.202004-1079oc

Figure Lengend Snippet: Figure 6. Loss of Lcn2 decreases T cell abundance and elevates pro-inflammatory cytokines during LUAD development.

Article Snippet: Following antigen retrieval at 100°C for 20 minutes (Bond ER Solution #1, Leica Biosystems), four-micron tissue sections were incubated with anti-LCN2 polyclonal goat primary antibody (AF1757, 1:1000 dilution, R&D Systems, Minneapolis, MN) and detected using the Bond Polymer Refine Detection kit (DS9800, Leica Biosystems).

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity

doi: 10.1016/j.omtn.2017.11.008

Figure Lengend Snippet: Response of Low-FBS/+FGF2 Cultured Astrocytes to LPS+IFN-γ Stimulation at the mRNA Level qRT-PCR quantification of relative mRNA expression levels of Ifngr1 , Ifngr2 , Ciita , Tlr4 , Gfap , Vim , Lcn2 , Nos2 , Tnf , Il1b , and Il6 . Expression relative to resting controls (2 −ΔΔCt method), Gapdh reference gene. Data expressed as the mean of n ≥ 3 biological replicates ± SD; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; or ****p ≤ 0.0001, relative to non-activated control samples (one-way ANOVA with Dunnett’s multiple comparison test).

Article Snippet: Anti-Lcn2 primary antibody (MAB1857; R&D Systems) was diluted (1:200) in a solution of PBS + 1% NGS ± 0.3% Triton X-100 and incubated at 4°C overnight.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Control, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity

doi: 10.1016/j.omtn.2017.11.008

Figure Lengend Snippet: Response of Low-FBS/+FGF2 Cultured Astrocytes to LPS+IFN-γ Stimulation at the Protein Level (A) Representative western blot illustrating temporal induction of pSTAT3 upon 1 and 48 hr of treatment with LPS+IFN-γ. (B and C) Representative western blots of activation markers in resting versus LPS+IFN-γ-stimulated low-FBS astrocytes: (B) Lcn2 expression in resting and 24-hr-stimulated astrocytes; and (C) GFAP and Vim levels under resting conditions and following 48 hr of activation followed by 3 or 5 days of rest. β-Actin was used as a loading control. (D) Representative immunofluorescence micrographs of GFAP, Vim, and Lcn2 immunoreactivity in resting and activated conditions (GFAP/Vim: green; Lcn2: red; DAPI nuclear stain: blue). Scale bars, 100 μm. (E) Secreted IL-6 as measured by ELISA, resting versus activated conditions, at 48 and 120 hr post-stimulation. Data are expressed as the mean of n ≥ 3 biological replicates ± SD. *p ≤ 0.05, relative to non-activated control samples (multiple t tests with statistical significance determined using the Holm-Sidak method, α = 0.05).

Article Snippet: Anti-Lcn2 primary antibody (MAB1857; R&D Systems) was diluted (1:200) in a solution of PBS + 1% NGS ± 0.3% Triton X-100 and incubated at 4°C overnight.

Techniques: Cell Culture, Western Blot, Activation Assay, Expressing, Control, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity

doi: 10.1016/j.omtn.2017.11.008

Figure Lengend Snippet: siRNA-3WJ Nanotherapeutics Specifically and Significantly Knock Down Expression of Target Genes (A) qPCR assays showing the knockdown of Gfap , Vim , and Lcn2 target mRNA by 5 nM doses of their respective siRNA-3WJ nanostructures. Downregulation measured 48 hr after transfection, under resting conditions. Mock (Lipofectamine-only) or non-targeted negative control 3WJs were used as negative controls. Gapdh was the reference gene. (B) Densitometric quantification of Vim (54 kDa) and GFAP (50 kDa) western blots showing siRNA-3WJ-induced protein knockdown in preventative activation conditions (20 DIV) and a representative image. Quantification is expressed relative to β-actin and normalized to non-transfected control samples. (C) Representative immunofluorescence micrographs depicting the decrease in GFAP and Vim immunoreactivity upon G10- or V10-3WJ treatment, respectively, in preventative activation conditions, compared with non-treated and N03-3WJ-treated controls (GFAP and Vim: green, DAPI-stained nuclei: blue). (D) Quantification and representative image of Lcn2 (23 kDa) western blot under resting , therapeutic , and preventative activation conditions upon L12-3WJ treatment (16 DIV), compared with non-treated controls. Quantification as per (B). (E) Lcn2 immunofluorescence, as per (C), with quantification of intensity density (n = 3 biological replicates, 2 coverslips per replicate, 10 fields of view per coverslip) of L12- and N03-3WJ-treated samples versus non-treated controls (Lcn2: red; DAPI-stained nuclei: blue). Scale bars, 100 μm. (F) Schematic of the LGV1-3WJ, a single 3WJ core functionalized with the siRNA moieties of each of the L12-, G10-, and V10-3WJs. In vitro transcribed RNA strands LGVa, LGVb, and LGVc are annealed together; black circles indicate a wobble base pair. (G) Agarose gel (3%, TBM buffer) illustrating the stepwise assembly and retarded mobility of the extended LGV1-3WJ construct relative to single- and double-stranded intermediates. Ultra-low dsDNA ladder is for reference. (H) qPCR assays showing the knockdown of Gfap , Vim , and Lcn2 target mRNA by a 5 nM dose of the LGV1-3WJ nanostructure, as per the single-siRNA 3WJs in (A). Quantitative results expressed as the mean of n ≥ 3 biological replicates ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; or ****p ≤ 0.0001, relative to control samples of the same activation condition (two-way ANOVA [qPCR] or one-way ANOVA [western blot] with Dunnett’s multiple comparison test).

Article Snippet: Anti-Lcn2 primary antibody (MAB1857; R&D Systems) was diluted (1:200) in a solution of PBS + 1% NGS ± 0.3% Triton X-100 and incubated at 4°C overnight.

Techniques: Knockdown, Expressing, Transfection, Negative Control, Western Blot, Activation Assay, Control, Immunofluorescence, Staining, In Vitro, Agarose Gel Electrophoresis, Construct, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity

doi: 10.1016/j.omtn.2017.11.008

Figure Lengend Snippet: L12-3WJ Treatment Ameliorates the Propagation of Classical Activation by Reactive Astrocytes (A) Schematic of the conditioned media transfer experiment. (B) ELISA quantification of Lcn2 in the supernatant media of resting or LPS+IFN-γ-stimulated astrocytes, as well as activated astrocytes treated with mock, N03, or L12 transfections, 48 hr after treatment. Asterisk (*) denotes statistical significance relative to the supernatant of non-transfected, activated astrocytes; number sign (#) is relative to non-transfected, resting astrocytes. ##p ≤ 0.01, ###p ≤ 0.001 relative to resting medium, ***p ≤ 0.001 relative to activated medium. (C–F) qPCR quantification of activation-associated genes in recipient cells 24 hr after transfer of media from treated astrocytes: (C) Gfap , (D) Vim , (E) Nos2 , and (F) Lcn2 ; results are expressed as the mean mRNA expression relative to those cells receiving conditioned media from activated controls (2 −ΔΔCt method) ( Gapdh reference gene). Data represent the mean of n ≥ 3 biological replicates ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; or ****p ≤ 0.0001, relative to control samples in the same activation condition (two-way ANOVA with Dunnett’s multiple comparison test).

Article Snippet: Anti-Lcn2 primary antibody (MAB1857; R&D Systems) was diluted (1:200) in a solution of PBS + 1% NGS ± 0.3% Triton X-100 and incubated at 4°C overnight.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Control, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity

doi: 10.1016/j.omtn.2017.11.008

Figure Lengend Snippet: 2′F-L12-3WJ Injection in an Experimental Model of Moderate Contusive Spinal Cord Injury Reduces Lcn2 mRNA and Protein Levels (A) Schematic representation of 2′F-L12-3WJ injection in an experimental model of moderate contusive spinal cord injury. (B) qPCR quantification showing the knockdown of Lcn2 in mice treated with 10 μg of 2′F-L12-3WJ, compared with injury-only control mice (sham), Lipofectamine-only treated mice, 10 μg of 2′F-N03-3WJ, and 1 μg of 2′F-L12-3WJ-treated mice (n = 3–4 mice per group). **p ≤ 0.01, relative to sham control mice (one-way ANOVA with Dunnett’s multiple comparison test). Data represent mean ± SD. (C) Quantification of Lcn2-postive volumes in injury-only control mice (sham) versus 10 μg of 2′F-L12-3WJ-treated mice. Data are expressed as % of Lcn2 + volume ± SD over the total volume (n = 4 mice per group). *p ≤ 0.05 (Student’s t test). Representative Lcn2 immunohistochemistry images in injury-only control (sham) and 10 μg of 2′F-L12-3WJ-treated mice, counterstained with hematoxylin. Original magnification ×4; scale bar, 500 μm. Corresponding representative 3D reconstructions depict the lesion volume in red, with blue contours representing Lcn2 + volumes and light gray contour representing the spinal cord.

Article Snippet: Anti-Lcn2 primary antibody (MAB1857; R&D Systems) was diluted (1:200) in a solution of PBS + 1% NGS ± 0.3% Triton X-100 and incubated at 4°C overnight.

Techniques: Injection, Knockdown, Control, Comparison, Immunohistochemistry