mouse anti human galectin3  (Proteintech)

Journal: Nature Communications

Article Title: Astrocytic phagocytosis contributes to demyelination after focal cortical ischemia in mice

doi: 10.1038/s41467-022-28777-9

Figure Lengend Snippet: A Representative images showing the changes of the reactive astrocytes (GFAP, red) containing MBP-positive (green) myelin inclusions (white arrows) in lysosome (LAMP1, cyan) among demyelination lesion. Scale bar, 20 μm. B Immunostaining images of triple-labeled astrocytes (GFAP + , red) containing dMBP-positive (green) myelin fragments in LAMP1 + lysosome (cyan). White arrows indicated phagocytic astrocytes. Scale bar, 20 μm. C , D Quantification of triple-positive astrocytes obtained from immunofluorescence staining ( n = 5 mice; adjusted ** P < 0.0017, *** P < 0.0002 vs. WT sham; # P < 0.0083, ### P < 0.0002 vs. WT dMCAO; two-way ANOVA, repeated-measures t -test). E Representative EM images of astrocytes in the ipsilateral CC of WT and Lcn2 −/− mice. Phagocytic inclusions with myelin-like structures were shown with red arrows. Scale bar, 5 μm. F Representative orthogonal views of Z-stack images obtained from two genotypes. GFAP + (red) and Galectin3 + (blue) astrocytes engulfed dMBP + (green) myelin debris after dMCAO. Scale bar, 20 μm. G Representative 3D images acquired from confocal images. Scale bar, 20 μm. H , I Quantifications showing the changes of co-positive (GFAP + and Galectin3 + ) phagocytic astrocytes and phagocytic astrocytes containing myelin debris (GFAP + , Galectin3 + and dMBP + ) ( n = 5 mice; mean ± S.D.; adjusted ** P < 0.0017, *** P < 0.0002 vs. WT sham; ### P < 0.0002 vs. WT dMCAO; two-way ANOVA, repeated-measures t -test). J Quantification of the number of myelin debris contacted with or internalized by a single astrocyte according to 3D reconstruction images ( n = 75 cells from five animals in each group; mean ± S.D.; adjusted *** P < 0.0002 vs. WT sham; ### P < 0.0002 vs. WT dMCAO; two-way ANOVA, repeated-measures t -test). Source data are provided as a Source Data file.

Article Snippet: Antibodies against Gbp2 (ab203238), GFAP (ab53554, ab7260), LCN2 (ab63929), LRP1 (ab92544), MAG (ab89780), MBP (ab40390), NF200 (ab7795), PLP (ab28486) and S100A10 (ab76472) were purchased from Abcam, UK; antibody against CD31 (550274) was purchased from BD Biosciences, USA; antibodies against GFAP (3670S), GFP (2955S, 2956S), p38 (9212S), pp38 (4511S) and β-actin (8457S) were purchased from Cell Signaling Technology, USA; antibodies against CC1 (OP80), dMBP (AB5864) and Olig2 (AB9610) were purchased from Millipore, USA; antibodies against C3d (AF2655), Galectin3 (AF1197), LAMP1 (AF4320) and LCN2 (AF1857) were purchased from R&D Systems, USA; antibodies against Gbp2 (sc-166960) and LRP1 (sc-57351) were purchased from Santa Cruz Biotechnology, USA; antibody against pLRP1 (PA5-101013) was purchased from Thermo Fisher, USA; antibody against Iba1 (019-19741) was purchased from Wako, Japan.

Techniques: Immunostaining, Labeling, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Astrocytic phagocytosis contributes to demyelination after focal cortical ischemia in mice

doi: 10.1038/s41467-022-28777-9

Figure Lengend Snippet: A Experimental flow chart. B Representative immunostaining images of the transfection of GFP (green) reporter LV into astrocytes (GFAP, red) with enforced cytosolic LCN2 (blue) expression in the CC. White arrows indicated astrocytes transfected with LV. Scale bar, 20 μm. C , D Representative images and statistical analysis of C3d (blue) co-stained with GFAP (red) in mice receiving LV-NC or LV- Lcn2 (Δ2–20) on the 3rd and 7th day after dMCAO ( n = 5 mice; mean ± S.D.; * P < 0.05, *** P < 0.001 vs. LV-NC 3d; two-way ANOVA, Tukey post hoc test). Scale bar, 20 μm. E – G Representative confocal, 3D images and quantifications of Galectin3 + (blue) phagocytic astrocytes (GFAP + , red) internalizing myelin debris (dMBP + , green). ( n = 75 cells from five animals in each group for quantification of debris in a single astrocyte, n = 5 mice for others; mean ± S.D.; ** P < 0.01, *** P < 0.001 vs. LV-NC 3d; # P < 0.05, ## P < 0.01 vs. LV- Lcn2 (Δ2–20) 3d; two-way ANOVA, Tukey post hoc test). Scale bar, 20 μm. H , I Representative MBP, MAG, NF200 staining and quantifications from Lcn2 −/− dMCAO mice receiving LV ( n = 5 mice; mean ± S.D.; ** P < 0.01, ***P < 0.001 vs. LV-NC 3d; two-way ANOVA, Tukey post hoc test). Scale bar, 20 μm. Source data are provided as a Source Data file.

Article Snippet: Antibodies against Gbp2 (ab203238), GFAP (ab53554, ab7260), LCN2 (ab63929), LRP1 (ab92544), MAG (ab89780), MBP (ab40390), NF200 (ab7795), PLP (ab28486) and S100A10 (ab76472) were purchased from Abcam, UK; antibody against CD31 (550274) was purchased from BD Biosciences, USA; antibodies against GFAP (3670S), GFP (2955S, 2956S), p38 (9212S), pp38 (4511S) and β-actin (8457S) were purchased from Cell Signaling Technology, USA; antibodies against CC1 (OP80), dMBP (AB5864) and Olig2 (AB9610) were purchased from Millipore, USA; antibodies against C3d (AF2655), Galectin3 (AF1197), LAMP1 (AF4320) and LCN2 (AF1857) were purchased from R&D Systems, USA; antibodies against Gbp2 (sc-166960) and LRP1 (sc-57351) were purchased from Santa Cruz Biotechnology, USA; antibody against pLRP1 (PA5-101013) was purchased from Thermo Fisher, USA; antibody against Iba1 (019-19741) was purchased from Wako, Japan.

Techniques: Immunostaining, Transfection, Expressing, Staining

Journal: Nature Communications

Article Title: Astrocytic phagocytosis contributes to demyelination after focal cortical ischemia in mice

doi: 10.1038/s41467-022-28777-9

Figure Lengend Snippet: A Experimental flow chart in vivo. B Representative fluorescent images of the transfection of GFP (green) reporter LV in astrocytes (GFAP, blue) with reduced LRP1 (red) in the CC of WT mice. White arrows indicated astrocytes transfected with lentiviruses. Scale bar, 20 μm. C , D Immunoblotting analyses and quantifications for LCN2, LRP1, pLRP1, p38 and pp38 in vivo ( n = 5 mice; mean ± S.D.; ** P < 0.01, *** P < 0.001 vs. LC-NC; paired t -test). Protein samples derived from the same experiment and gels/blots were processed in parallel. E , F Representative images and quantifications showing the expression of LCN2 (red) and C3d (red) in astrocytes (GFAP, green) after dMCAO ( n = 5 mice; mean ± S.D.; paired t -test). Scale bar, 20 μm. G – J Representative confocal, 3D reconstruction images and quantifications of phagocytic astrocytes (Galectin3 + , blue; GFAP + , red) engulfing dMBP + (green) debris in mice receiving LC-NC or LV- Lrp1 -RNAi. ( n = 75 cells from five animals in each group for quantification of debris in single astrocyte, n = 5 mice for others; mean ± S.D.; * P < 0.05, *** P < 0.001 vs. LC-NC; paired t -test). Scale bar, 20 μm. K Experimental flow chart in vitro. L Representative immunocytochemistry images of GFAP (green), LRP1 (red), LCN2 (blue) and DAPI (cyan) after lentiviruses transfection. Scale bar, 20 μm. M – P Representative confocal images, ELISA and flow cytometry analyses showing the differences of astrocytic phagocytosis between the LC-NC and LV- Lrp1 -RNAi groups in vitro ( n = 5 independent primary cell cultures; mean ± S.D.; * P < 0.05 vs. LC - NC; paired t -test). Scale bar, 20 μm. In the box plots ( N , P ), the middle bar represents the median, the box represents the interquartile range and whiskers indicate the maximum and minimum values. Dots are all the data points. Source data are provided as a Source Data file.

Article Snippet: Antibodies against Gbp2 (ab203238), GFAP (ab53554, ab7260), LCN2 (ab63929), LRP1 (ab92544), MAG (ab89780), MBP (ab40390), NF200 (ab7795), PLP (ab28486) and S100A10 (ab76472) were purchased from Abcam, UK; antibody against CD31 (550274) was purchased from BD Biosciences, USA; antibodies against GFAP (3670S), GFP (2955S, 2956S), p38 (9212S), pp38 (4511S) and β-actin (8457S) were purchased from Cell Signaling Technology, USA; antibodies against CC1 (OP80), dMBP (AB5864) and Olig2 (AB9610) were purchased from Millipore, USA; antibodies against C3d (AF2655), Galectin3 (AF1197), LAMP1 (AF4320) and LCN2 (AF1857) were purchased from R&D Systems, USA; antibodies against Gbp2 (sc-166960) and LRP1 (sc-57351) were purchased from Santa Cruz Biotechnology, USA; antibody against pLRP1 (PA5-101013) was purchased from Thermo Fisher, USA; antibody against Iba1 (019-19741) was purchased from Wako, Japan.

Techniques: In Vivo, Transfection, Western Blot, Derivative Assay, Expressing, In Vitro, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: eLife

Article Title: The role of P2Y12 in the kinetics of microglial self-renewal and maturation in the adult visual cortex in vivo

doi: 10.7554/eLife.61173

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Galectin3 (Goat polcylonal) , R and D systems , RRID: AB_2234687 Cat# AF1197 , IF(1:1500).

Techniques: Generated, Adhesive, Microscopy, Software

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 is increased in human and mouse AD brains and marks a microglial phenotype associated with Aβ plaques. a Western blot analyses of cortex from human AD cases ( n = 6) and age-matched healthy controls ( n = 5). b Galectin-3 (gal3) staining mainly coincided with Iba1 + microglia found around Aβ plaques in human AD brains. c Immunohistochemistry showed high levels of gal3 in microglia in AD brains, as compared to Iba1-staining (low levels of gal3 was detected in association to blood vessels). d Gal3 protein was significantly upregulated in the cortex of 5xFAD mice in a time-dependent fashion (WT, 6 months old). e Gal3 expression was found in Iba1 + cells around Αβ plaques. Statistical significance was calculated by one-way ANOVA with Tukey’s correction ( d ) or Student’s t test ( a ). *p < 0.05; **p < 0.01. Data are shown as mean ± SD. The human AD cases are described in suppl. Tables S1 and S2 (online resources 8 and 9)

Article Snippet: For single immunolabeling, sections were immunoreacted with one of the following primary antibodies: anti-Aβ42 rabbit polyclonal (1:5000 dilution, Abcam), anti-amyloid precursor protein (APP) rabbit polyclonal (1:20,000 dilution, Sigma), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500 dilution, AbD Serotec), anti-galectin3 (gal3) goat polyclonal (1:3000 dilution, R&D) over 24 or 48 h at room temperature.

Techniques: Western Blot, Staining, Immunohistochemistry, Expressing

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 deficiency/inhibition reduces the microglial inflammatory response in vitro. a Reduced cytokine levels in culture medium from Gal3KO primary microglial cultures compared to WT after fΑβ treatment for 12 h. b WT primary microglial cultures increase the release of gal3 upon stimulation with fAβ. In vitro experiments represent a minimum of three independent experiments. Statistical significance was calculated by Student’s t-test ( b ), or one-way ANOVA with Tukey’s correction ( a ) *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SD

Article Snippet: For single immunolabeling, sections were immunoreacted with one of the following primary antibodies: anti-Aβ42 rabbit polyclonal (1:5000 dilution, Abcam), anti-amyloid precursor protein (APP) rabbit polyclonal (1:20,000 dilution, Sigma), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500 dilution, AbD Serotec), anti-galectin3 (gal3) goat polyclonal (1:3000 dilution, R&D) over 24 or 48 h at room temperature.

Techniques: Inhibition, In Vitro

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 colocalizes with TREM2. a , b Iba1 + cells expressing galectin-3 (gal3) around Aβ plaque in 5xFAD mice. c Reduced number of Iba1 + microglial cells around Αβ plaques in 5xFAD/Gal3KO mice compared to 5xFAD mice (% of Αβ area). d Number of Iba1 + cells expressing gal3 in 5xFAD (% of Αβ area). e , f Gal3 and TREM2 in plaque-associated microglia in the brain of 5xFAD mice reveals colocalization of gal3 and TREM2. g Gal3 and TREM2 colocalization in 5xFAD mouse brain using STORM microscopy. Statistical significance was calculated by Student’s t test. *p < 0.05. Data are shown as mean ± SEM. All images were taken in 5xFAD mice at 18 months

Article Snippet: For single immunolabeling, sections were immunoreacted with one of the following primary antibodies: anti-Aβ42 rabbit polyclonal (1:5000 dilution, Abcam), anti-amyloid precursor protein (APP) rabbit polyclonal (1:20,000 dilution, Sigma), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500 dilution, AbD Serotec), anti-galectin3 (gal3) goat polyclonal (1:3000 dilution, R&D) over 24 or 48 h at room temperature.

Techniques: Expressing, Microscopy

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 interacts with TREM2 through its carbohydrate-binding domain. a Fluorescent anisotropy assay for galectin-3 (gal3)/TREM2 interaction. Data are presented as % of TREM2–gal3 binding (gal3, WT and mutant gal3 with deficient carbohydrate-binding domain, R186S) and fluorescent probe interaction, by increasing concentrations of TREM2, together with the calculated K d values for the gal3/TREM2 interaction ( n = 2). b Control and DAP12 reporter cell lines were stimulated with increasing concentrations of gal3 (250 nM–2.5 µM), ionomycin and phosphatidylserine (PS). Statistical significance was calculated by Student’s t test or one-way ANOVA with Bonferroni’s post hoc test. *p < 0.05; **p < 0.01. Data are shown as mean ± SD

Article Snippet: For single immunolabeling, sections were immunoreacted with one of the following primary antibodies: anti-Aβ42 rabbit polyclonal (1:5000 dilution, Abcam), anti-amyloid precursor protein (APP) rabbit polyclonal (1:20,000 dilution, Sigma), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500 dilution, AbD Serotec), anti-galectin3 (gal3) goat polyclonal (1:3000 dilution, R&D) over 24 or 48 h at room temperature.

Techniques: Binding Assay, Mutagenesis, Control

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 induces the formation of insoluble Αβ aggregates following injections of Αβ monomers in the hippocampi of WT mice. Αβ monomers were injected with galectin-3 (Gal3) (Aβ + Gal3) or without (Aβ) after 1 h Aβ monomers incubation w/o gal3 into the right or left hippocampi of WT mice, respectively. a Staining for Αβ and gal3 in the left hippocampus (only Aβ monomers injected). b , c Staining for Αβ and gal3 in the right hippocampus (Aβ + gal3 injected). Dashed frames in b are magnified and shown in c . d Thioflavin-S and Αβ staining of the left hippocampus (only Aβ injected). e Thioflavin-S and Αβ staining of the right hippocampus (Aβ + gal3 injected). White arrows point to gal3 and thioflavin-S + aggregates. f , g Iba1 and GFAP immunoreactivity in the right hippocampus (Αβ + gal3 were injected)

Article Snippet: For single immunolabeling, sections were immunoreacted with one of the following primary antibodies: anti-Aβ42 rabbit polyclonal (1:5000 dilution, Abcam), anti-amyloid precursor protein (APP) rabbit polyclonal (1:20,000 dilution, Sigma), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500 dilution, AbD Serotec), anti-galectin3 (gal3) goat polyclonal (1:3000 dilution, R&D) over 24 or 48 h at room temperature.

Techniques: Injection, Incubation, Staining