ccl2 neutralising antibody  (R&D Systems)

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A Proteome profiler analysis of 111 inflammatory proteins in gastrocnemius (GC), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles and spinal cord (SC) samples from late symptomatic (postnatal day 19) hTDP-43 Tg/Tg mice. Fold change in protein levels is presented relative to wild-type (WT) littermate controls. In the GC muscle, CCL2, CCL3, CCL4, CCL5 and CCL6 chemokines showed the greatest increase in protein levels (magnified and highlighted in the top right panel). Exact fold changes are available in the Source Data file. B –E Quantitative analysis of CCL2 ( B ), CCL3 ( C ), CCL4 ( D ) and CCL5 ( E ) proteins in the GC and TA muscles and SC using Legendplex immunoassay. Data is presented as mean ± sem. The Legendplex multiplex immunoassay and statistical analysis was performed with n = 4 mice in the GC, TA and SC of the WT group and TA and SC of the Tg/Tg group. The GC of the Tg/Tg group was quantified in n = 3 mice. Data analysis: In each case ( B – E ), unpaired, one-tailed t-test was performed for direct comparison of WT vs Tg/Tg groups; ( B) gastrocnemius : p = 0.0350; ( C) g astrocnemius : p = 0.0174; ( C) tibialis anterior: p = 0.0318; ( C) spinal cord: p = 0.0083; ( D) gastrocnemius: p = 0.0209; ( E) gastrocnemius: p = 0.0031; ( E) tibialis anterior: p = 0.0456. * = p < 0.05; ** = p < 0.01.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Muscles, Multiplex Assay, One-tailed Test, Comparison

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A–C CCL2 staining (green) in the GC muscle of wild-type (WT; A ) and late symptomatic postnatal day 19 (P19) hTDP-43 Tg/Tg (Tg/Tg; B, C ) mice. Arrows indicate CCL2 + cells in the close vicinity of neuromuscular junctions (NMJs), visualised with α-bungarotoxin (BTX; magenta). C High magnification image of CCL2 + cells around a single NMJ in the GC muscle of late stage hTDP-43 Tg/Tg mice. Innervation is shown through NEFH/SV2a (gray) staining. Asterisks label individual nuclei of CCL2 + cells. 3D-reconstructed version of this image is available as Supplementary movie . D–F Quantitative analysis of the ratio of NMJs with perisynaptic CCL2-expressing cells in presymptomatic ( D ), early symptomatic ( E ) and late symptomatic ( F ) mice. Data is presented as mean ± sem. Scale bar represents 50 µm on ( A, B ) and 10 µm on ( C ). Data analysis: ( D ) Presymptomatic stage (postnatal day 7): n = 4 (WT), n = 3 (Tg/Tg), unpaired, two-tailed t-test, p = 0.0466; ( E ) Early symptomatic stage (postnatal day 15): n = 3 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0088; ( F ) Late symptomatic stage: n = 4 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0011. * = p < 0.05; ** = p < 0.01.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

doi: 10.1038/s41467-025-62351-3

Figure Lengend Snippet: A Schematic of experimental design. B–D Representative images of NMJ denervation in the gastrocnemius (GC) muscle of untreated ( B ), IgG-treated ( C ) and CCL2-neutralising antibody-treated ( D ) late symptomatic postnatal day 19 (P19) hTDP-43 Tg/Tg mice. Red arrows indicate fully innervated NMJs, as revealed by the presynaptic NEFH/SV2a staining. Scale bar represents 40 µm. E–G Representative images of the CD45 + leukocyte infiltration in the innervation zone of the GC muscle in untreated ( E ), IgG-treated ( F ) and CCL2-neutralising antibody-treated ( G ) late symptomatic hTDP-43 Tg/Tg mice. Scale bar represents 40 µm. H Qualitative analysis of NMJ innervation, representing the ratio of fully innervated NMJs in the GC muscle. Analysis was performed with n = 7 (untreated) and n = 4 (IgG, CCL2) mice/group and one-way ANOVA with Fisher’s LSD post-hoc, no treatment vs. IgG: p < 0.0001, no treatment vs. CCL2: p < 0.0001. On average 30-40 NMJs were analysed per muscle. I–K Quantitative morphometric analysis of the NMJs revealed improvement in axonal diameter ( I ), nerve terminal area ( J ) and overlap ( K ) variables. The exact p values and statistical analyses are detailed in Table . L, M Quantitative analysis of CD45 + ( L ) and CCR2 + ( M ) cell count in the late symptomatic GC muscle of hTDP-43 Tg/Tg mice. Analysis was performed on n = 8 (untreated) and n = 4 (IgG, CCL2) mice/group. Data analysis: ( L) CD45 analysis: Kruskal-Wallis test and uncorrected Dunn’s post-hoc [Shapiro-Wilk normality test, ALS untreated group: p = 0.0091, ALS IgG group: p = 0.7802, ALS CCL2 group p = 0.3820], no treatment vs. IgG: p = 0.0114, no treatment vs. CCL2: p = 0.0053; ( M) CCR2 analysis: one-way ANOVA with Fisher’s LSD post-hoc, no treatment vs. IgG: p < 0.0001, no treatment vs. CCL2: p = 0.0002. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data is presented as mean ± sem.

Article Snippet: To assess the effects of local immunomodulatory treatment on NMJ denervation and immune cell infiltration, hTDP-43 Tg/Tg mice received two intramuscular injections (at P7 and P14) of either CCL2 neutralising antibody [Cat# AF-479-NA, RRID: AB_354500, R&D Systems, Minneapolis, MN, USA; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in phosphate buffer saline (PBS)] or isotypic normal goat IgG (Cat# AB-108-C, RRID:AB_354267, R&D Systems; 0.4 mg/kg body weight, at the concentration of 0.2 mg/ml, diluted in PBS) in the severely affected GC muscle.

Techniques: Staining, Cell Counting

Journal: Poultry Science

Article Title: Prolactin promotes transepithelial migration of lymphocytes through CCL2

doi: 10.1016/j.psj.2025.105322

Figure Lengend Snippet: (A) The co-culture setup of mammary epithelial cells and T lymphocytes. (B) The number of lymphocytes in the lower chamber was counted by a cell counter, ** P < 0.01; (C) Lymphocytes across breast epithelial cells were observed by fluorescence confocal microscopy; the left and right images are the cross-section and a vertical view of Transwell chamber, respectively. (D) The number of migrated lymphocytes under prolactin stimulation with or without CCL2 neutralization was quantified using a cell counter. CCL2 neutralizing antibody was added to the lower chamber simultaneously with prolactin, ** P < 0.01; (E) Lymphocytes across breast epithelial cells were observed by fluorescence confocal microscopy; the left and right images are the cross-section and a vertical view of the Transwell chamber, respectively.

Article Snippet: One group was treated with 50 ng/mL prolactin (1445-PR, R&D Systems) and 5 μg/mL CCL2 neutralizing antibodies (goat anti-mouse CCL2, AF-479-NA, R&D Systems) to block CCL2 ( ).

Techniques: Co-Culture Assay, Fluorescence, Confocal Microscopy, Neutralization

Journal: Cell communication and signaling : CCS

Article Title: IL-23 promotes neuronal ferroptosis via IL-23R/STAT3 signaling after traumatic brain injury.

doi: 10.1186/s12964-025-02319-4

Figure Lengend Snippet: Fig. 1 Expression of IL-23 and IL-23R in human TBI patients. A: ELISA analysis of serum IL-23 levels in TBI patients (n = 43) and healthy controls (n = 7). Data are presented as mean ± SD. ****p < 0.0001. B: Representative immunohistochemical images of IL-23 in cortical tissue from control and TBI patients, with quantification of IL-23-positive area (mm2) (C). Data are presented as mean ± SD (n = 6 subjects/group). ****p < 0.0001. Scale bar = 50 μm. D: Representa tive immunohistochemical images of IL-23R in cortical tissue from control and TBI patients, with quantification of IL-23R-positive area (mm2) (E). Data are presented as mean ± SD (n = 6 subjects/group). ****p < 0.0001. Scale bar = 50 μm. F: Representative images of quadruple immunofluorescence staining showing colocalization of NeuN (red), IL-23R (green), CCL2(yellow) and nuclear DAPI (blue) in human cortical tissue. Scale bar = 50 μm. G: Quantification of NeuN+IL-23R+ cells per mm². Data are presented as mean ± SD (n = 6 subjects/group). ****p < 0.0001

Article Snippet: The following primary antibodies were applied overnight at 4 °C: rabbit anti-GFAP (1:200, 3670 S, Cell Signaling Technology, USA), rabbit anti-MPO (1:200, ab208670, Abcam, UK), goat anti-CD31 (1:100, AF806, R&D Systems, USA), rabbit anti-JAK2 (1:200, A19629, ABclonal Technology, China), rabbit anti-CCR2 (1:200, ab313463, Abcam, UK), rabbit anti-ACSL4 (1:200, ab155282, Abcam, UK), rabbit anti-phospho-STAT3 (Tyr705) (1:100, 9145s, Cell Signaling Technology, USA), rabbit anti-IL-23R (1:200, A1613, ABclonal Technology, China), rat anti-Macrophage ([RM0029-11H3], 1:100, ab56297, Abcam, UK), mouse anti-IL-23 (1:100, sc-271279, Santa Cruz Biotechnology, USA), anti-NeuN antibody ([EPR12763], 1:200, ab177487, Abcam, UK), and goat anti-CCL2 antibody (1:200, AF-479-NA, R&D Systems, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Control, Immunofluorescence, Staining

Journal: Cell communication and signaling : CCS

Article Title: IL-23 promotes neuronal ferroptosis via IL-23R/STAT3 signaling after traumatic brain injury.

doi: 10.1186/s12964-025-02319-4

Figure Lengend Snippet: Fig. 6 CCL2 expression patterns and colocalization with IL-23R in human and mouse TBI. A: Double immunofluorescence showing CCL2 (red) colo calization with cell-type specific markers (green) in cortical sections at 3 days post-TBI and sham controls. Cell markers include: NeuN (neurons), CD31 (endothelial cells), RM0029-11H3 (macrophages), GFAP (astrocytes), and IBA1 (microglia). DAPI counterstain in blue. Scale bar = 50 μm. B: Four-channel immunofluorescence of human cortical tissue showing NeuN (red), CCL2 (magenta), IL-23R (green) and DAPI (blue) in control and TBI specimens. Scale bar = 50 μm. C: Similar staining in mouse cortical sections showing differential expression patterns between sham and TBI groups. Scale bar = 50 μm

Article Snippet: The following primary antibodies were applied overnight at 4 °C: rabbit anti-GFAP (1:200, 3670 S, Cell Signaling Technology, USA), rabbit anti-MPO (1:200, ab208670, Abcam, UK), goat anti-CD31 (1:100, AF806, R&D Systems, USA), rabbit anti-JAK2 (1:200, A19629, ABclonal Technology, China), rabbit anti-CCR2 (1:200, ab313463, Abcam, UK), rabbit anti-ACSL4 (1:200, ab155282, Abcam, UK), rabbit anti-phospho-STAT3 (Tyr705) (1:100, 9145s, Cell Signaling Technology, USA), rabbit anti-IL-23R (1:200, A1613, ABclonal Technology, China), rat anti-Macrophage ([RM0029-11H3], 1:100, ab56297, Abcam, UK), mouse anti-IL-23 (1:100, sc-271279, Santa Cruz Biotechnology, USA), anti-NeuN antibody ([EPR12763], 1:200, ab177487, Abcam, UK), and goat anti-CCL2 antibody (1:200, AF-479-NA, R&D Systems, USA).

Techniques: Expressing, Immunofluorescence, Control, Staining, Quantitative Proteomics

Journal: Cell communication and signaling : CCS

Article Title: IL-23 promotes neuronal ferroptosis via IL-23R/STAT3 signaling after traumatic brain injury.

doi: 10.1186/s12964-025-02319-4

Figure Lengend Snippet: Fig. 7 CCL2/CCR2 signaling regulates IL-23 expression in macrophages after TBI. A: Four-channel immunofluorescence of human cortical tissue showing RM0029-11H3 (magenta), CCR2 (green), IL-23 (red) and DAPI (blue) in control and TBI specimens. Scale bar = 50 μm. B: Similar staining pattern in mouse cortical sections comparing sham and TBI groups. Scale bar = 50 μm. C: Left: Representative Western blots showing CCR2, CCL2, and IL-23 expression after anti-CCL2 treatment. Right: Densitometric analysis normalized to β-actin. n = 6/group, *p < 0.05, ***p < 0.001. D, E: Double immunofluorescence showing RM0029-11H3 (macrophages, green) with CCR2 (D) or IL-23 (E) (red) at 3 days post-TBI. Enlarged views of boxed regions shown at right. Adjacent graphs show quantification of positive cells per mm2. n = 6/group, ****p < 0.0001. DAPI counterstain in blue. Scale bar = 50 μm

Article Snippet: The following primary antibodies were applied overnight at 4 °C: rabbit anti-GFAP (1:200, 3670 S, Cell Signaling Technology, USA), rabbit anti-MPO (1:200, ab208670, Abcam, UK), goat anti-CD31 (1:100, AF806, R&D Systems, USA), rabbit anti-JAK2 (1:200, A19629, ABclonal Technology, China), rabbit anti-CCR2 (1:200, ab313463, Abcam, UK), rabbit anti-ACSL4 (1:200, ab155282, Abcam, UK), rabbit anti-phospho-STAT3 (Tyr705) (1:100, 9145s, Cell Signaling Technology, USA), rabbit anti-IL-23R (1:200, A1613, ABclonal Technology, China), rat anti-Macrophage ([RM0029-11H3], 1:100, ab56297, Abcam, UK), mouse anti-IL-23 (1:100, sc-271279, Santa Cruz Biotechnology, USA), anti-NeuN antibody ([EPR12763], 1:200, ab177487, Abcam, UK), and goat anti-CCL2 antibody (1:200, AF-479-NA, R&D Systems, USA).

Techniques: Expressing, Immunofluorescence, Control, Staining, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: IL-23 promotes neuronal ferroptosis via IL-23R/STAT3 signaling after traumatic brain injury.

doi: 10.1186/s12964-025-02319-4

Figure Lengend Snippet: Fig. 8 Anti-CCL2 Treatment Reduces Neuronal Death and Improves Functional Recovery after TBI. A-D: Neuronal death assessment in ipsilateral cortex at 3 days post-TBI. Immunofluorescence showing NeuN (red) and TUNEL (green) with enlarged views of boxed regions (A). FJC staining (green) with enlarged views of boxed regions (B). Quantification of TUNEL+ neurons (C) and FJC+ cells (D). Values shown as mean ± SD, n = 6 per group. ****p < 0.0001. Nuclei counterstained with DAPI (blue). Scale bar = 50 μm. E-H: Behavioral assessment at days 1 and 3 post-TBI. Rotarod performance (E, G) and mNSS scores (F, H). Data are shown as mean ± SD. Sham and Sham + Anti-CCL2 groups: n = 6; TBI group: n = 10 on day 1, n = 9 on day 3 (due to one animal death); TBI + Anti-CCL2 group: n = 8. *p < 0.05, ****p < 0.0001

Article Snippet: The following primary antibodies were applied overnight at 4 °C: rabbit anti-GFAP (1:200, 3670 S, Cell Signaling Technology, USA), rabbit anti-MPO (1:200, ab208670, Abcam, UK), goat anti-CD31 (1:100, AF806, R&D Systems, USA), rabbit anti-JAK2 (1:200, A19629, ABclonal Technology, China), rabbit anti-CCR2 (1:200, ab313463, Abcam, UK), rabbit anti-ACSL4 (1:200, ab155282, Abcam, UK), rabbit anti-phospho-STAT3 (Tyr705) (1:100, 9145s, Cell Signaling Technology, USA), rabbit anti-IL-23R (1:200, A1613, ABclonal Technology, China), rat anti-Macrophage ([RM0029-11H3], 1:100, ab56297, Abcam, UK), mouse anti-IL-23 (1:100, sc-271279, Santa Cruz Biotechnology, USA), anti-NeuN antibody ([EPR12763], 1:200, ab177487, Abcam, UK), and goat anti-CCL2 antibody (1:200, AF-479-NA, R&D Systems, USA).

Techniques: Functional Assay, Immunofluorescence, TUNEL Assay, Staining