goat anti mouse gal3 antibody  (R&D Systems)

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: Antibodies used for immunostainings

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques:

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: Antibodies used for western blot

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Western Blot

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: Galectin-3 (GAL3) is associated with Lewy Bodies (LB) and Pale Bodies (PB) in PD patients. a Immunofluorescence analysis of GAL3 in association with distinct forms of human α-synuclein (hSYN) aggregation. Β-sheet structure marker Methoxy-X04 was used to discriminate between LB and PB. Multiple core LB and PB are shown. GAL3 is present in both types of aggregates independently of neuromelanin presence. Scale bar 10 µm. b GAL3 is present in a diverse subset of hSYN accumulations with a precise negative correlation (blue arrows). Scale bar 10 µm. c Proportion of hSYN aggregates that are associated with GAL3. Methoxy-X04 was used as a specific marker of LB. Single (sLB) and multiple core LB (mLB) were discriminated ( p < 0.05). d Protein levels of GAL3 measured by ELISA in the Cortex of Control and PD Patients (PD-Cx) ( p < 0.001), and in the Substantia nigra (PD-SN) of PD patients

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Immunofluorescence, Marker, Enzyme-linked Immunosorbent Assay

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: GAL3 variably associates with lysosomes in the outer layers LB in all the studied patients. a GAL3 surrounding LB was found in all 6 patients studied (P.1–6). Variable amount of GAL3 vesicles was found. Note lower hSYN staining in the presence of GAL3. Scale bar 10 µm. b High resolution microscopy showed a ring-like pattern for GAL3 without any hSYN inside. Scale bar 10 µm. c Immunofluorescence analysis revealed that GAL3 is associated with recruited lysosomes (LAMP1) in the vicinities of LB. Scale bar 10 µm. d Combination of GAL3 immunohistochemistry with immunofluorescence showed that GAL3 is associated with autofluorescent lipofuscin vesicles in PD patients. Scale bar 10 µm. e Immunofluorescence analysis revealed that GAL3 accumulates inside MAP2 + neurons in the viccinities of LB. Scale bar 10 µm

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Staining, Microscopy, Immunofluorescence, Immunohistochemistry

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: Recombinant galectin-3 (Gal3) impairs synuclein aggregation in vitro. a Thioflavin-T (ThT) aggregation assay showed a rapid aggregation for recombinant human α-synuclein (αSyn) that was impaired in the presence of recombinant Gal3 (purple line). Notably, carbohydrate recognition domain (CRD) mutation (Gal3 R186S ) reverted this effect. b Proteinase K (PK) digestion at increasing concentration of resultant conditions from a) showed a lower stability in the presence of Gal3 (red line). c When Gal3 was added to αSyn pre-formed fibrils (PFF) after aggregation was completed, an increased signal was observed in the presence of ThT after 15 h. d PK digestion at increasing concentration of resultant fibrils from ( c ) showed similar stability of PFF in the presence of Gal3. e Electron microscopy images after uranyl negative staining of PFF after 24 h incubation with Gal3 (right panels). Note a marked disorganization of the fibrils network after Gal3 incubation with increased shortened species (upper right panel), and the change of morphology (lower right panel) with rounded structures attached to the fibrils. Scale bar 1 µm (upper panels) and 200 nm (lower panels). f Native PAGE Western Blot of the final results obtained in c ) Note that Gal3 promoted an increase in smaller soluble species released by αSyn fibrils. g Direct interaction of Gal3 with different αSyn species was investigated by ELISA. 2 µM Gal3 concentration were precoated in a 96 well plate and 2 µM αSyn species were incubated. 450 nm absorbance was measured to detect bounded protein. All types of species presented high affinity for Gal3 coated well compared with the control condition in absence of αSyn ( p < 0.001). No relevant absorbance was detected in the absence of precoated Gal3 (data not shown). h Addition of sonicated PFF pre-incubated with gal3 (PFFgal3) for 30 min to dopaminergic cell line N27 for 48 h led to a decreased number of cells compared with PFF alone (** p < 0.01; *** p < 0.001). i Graphical abstract representing the hypothesis proposed based on our in vitro studies about Gal3-αSyn interaction. Gal3 could impact αSyn elongation in de novo formation of fibrils while also affecting structured fibrils with little impact on the dense core but release of small species

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Recombinant, In Vitro, Mutagenesis, Concentration Assay, Electron Microscopy, Negative Staining, Incubation, Clear Native PAGE, Western Blot, Enzyme-linked Immunosorbent Assay, Sonication

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: GAL3 early overexpression leads to chronic activation and neuronal internalization. a Western Blot against GAL3 from brain homogenates from WT and Gal3KO mice revealed constitutive expression of GAL3 in WT mice. b Western Blot quantification of total GAL3 protein in WT mesencephalon samples. No difference was found between contralateral (Right hemisphere, RH) and ipsilateral (Left hemisphere, LH) hemispheres. Data are expressed as percentage fold to actin. c Double immunofluorescence 6 months after adenovirus injection showed clusters of CD11B + microglial cells highly reactive for GAL3. Internalized pSYN led to overexpression of GAL3 in WT microglia. pSYN was internalized by microglia independently of GAL3 genotype. Scale bar 20 µm. d TNFα quantification in SN and STR was performed on a MesoScale Discovery platform analysing brain extracts from AAV5-hSYN injected SN and STR ( p < 0.05). e Neuronal primary cell culture from WT mice showed efficient Gal3 internalization after incubation with 0.8 µM gal3 for 10 days. Note no difference in endogenous αSyn staining after addition of Gal3. f hSYN/GAL3 double immunofluorescence from injection area of mice WT brains 2 weeks after injection revealed no colocalization and significant upregulation of GAL3. Scale bar 50 µm. GAL3 lo /hSYN colocalization (white arrow) can be found near highly reactive GAL3 + cell indicating GAL3 release and neuronal GAL3 internalization. Scale bar 10 µm. g hSYN/GAL3 double immunofluorescence of mice WT brains 4 weeks after adenovirus injection revealed neuronal GAL3 staining. Scale bar 10 µm

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Over Expression, Activation Assay, Western Blot, Expressing, Immunofluorescence, Injection, Cell Culture, Incubation, Staining

Journal: In vivo (Athens, Greece)

Article Title: Expression of Galectin 3 and Activating Transcription Factor 3 in Nigral Dopaminergic Neurons of 6-Hydroxydopamine Induced Parkinsonian Rat Model.

doi: 10.21873/invivo.13938

Figure Lengend Snippet: Figure 2. Gal3 expression was upregulated in the ipsilateral nigral dopaminergic neurons. (A) Photographs of Gal3 immunohistochemistry in the contralateral (Cont) and ipsilateral (Ispi) substantia nigra (SN) after 6‐hydroxydopamine (6‐OHDA) lesion. Gal3 immunoreactivity was upregulated in the ipsilateral SN. (B) High magnification image of the area designated by the black square inside of (A). Morphologically typical multipolar neurons with large cell body and many prominent processes (arrows) and neuroglia with small cell bodies and many long and slender radiating processes (arrowheads) were positive for Gal3 in the ipsilateral SN. (C) Double immunofluorescence images of Gal3 and TH and their merged image, in the ipsilateral SN at 5 dpl. Gal3 was expressed in many nigral dopaminergic neurons (arrows). (D) Double immunofluorescence images of Gal3 and Iba1 and their merged image. Gal3 was also expressed in many microglial cells (arrows). (E) Double immunofluorescence images of Gal3 and GFAP and their merged image. None of the cells immunolabeled for Gal3 were colocalized with GFAP‐positive astrocytes (arrows). (F) Quantitative analysis showing the average number of neurons expressing Gal3 per unit area (mm2) at each time point. Scale bars represent 200 μm in (A), 50 μm in (B and E). Data are expressed as means±SEM (n=6, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). dpl, Days post‐lesion.

Article Snippet: Antibody Catalog Manufacturer Host Description Concentration number IHC IF Gal3 AF‐1197 R&D system Goat Wide range of cell type 1:500 1:50 ATF3 Sc‐188 Santa Cruz Rabbit Wide range of cell type 1:1000 1:100 TH MAB5280 Millipore Mouse Tyrosine hydroxylase 1:1000 1:500 Iba1 AB283319 Abcam Mouse Microglia marker 1:200 GFAP AB279289 Abcam Mouse Astrocyte marker 1:200 FG AB153 Millipore Rabbit Fluorogold 1:500 1:100 Gal3, Galectin 3; ATF3, activating transcription factor 3; TH, tyrosine hydroxylase; Iba1, ionized calcium‐binding adapter molecule 1; GFAP, glial fibrillary acidic protein; FG, fluorogold; IHC, immunohistochemistry; IF, immunofluorescence. antibodies against TH (1:500), or rabbit polyclonal antibodies against FG (1:100).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Immunolabeling

Journal: In vivo (Athens, Greece)

Article Title: Expression of Galectin 3 and Activating Transcription Factor 3 in Nigral Dopaminergic Neurons of 6-Hydroxydopamine Induced Parkinsonian Rat Model.

doi: 10.21873/invivo.13938

Figure Lengend Snippet: Figure 4. Co‐localization of Gal3 and ATF3 in the 6‐OHDA insulted dopaminergic neurons. (A) Representative photomicrographs showing triple immunofluorescence labeling for FG, Gal3, and ATF3 and their merged image in the ipsilateral SN after 6‐OHDA lesion at 5 dpl. (B) High magnification image of the area designated by the white square inside of (A). ATF3 and Gal3 were colocalized in the dopaminergic neurons that are labeled with FG, i.e., neurons that have been retrogradely insulted with 6‐OHDA. Scale bar represents 200 μm and 50 μm in (A) and (B), respectively. dpl, Days post‐lesion; Ipsi, ipsilateral.

Article Snippet: Antibody Catalog Manufacturer Host Description Concentration number IHC IF Gal3 AF‐1197 R&D system Goat Wide range of cell type 1:500 1:50 ATF3 Sc‐188 Santa Cruz Rabbit Wide range of cell type 1:1000 1:100 TH MAB5280 Millipore Mouse Tyrosine hydroxylase 1:1000 1:500 Iba1 AB283319 Abcam Mouse Microglia marker 1:200 GFAP AB279289 Abcam Mouse Astrocyte marker 1:200 FG AB153 Millipore Rabbit Fluorogold 1:500 1:100 Gal3, Galectin 3; ATF3, activating transcription factor 3; TH, tyrosine hydroxylase; Iba1, ionized calcium‐binding adapter molecule 1; GFAP, glial fibrillary acidic protein; FG, fluorogold; IHC, immunohistochemistry; IF, immunofluorescence. antibodies against TH (1:500), or rabbit polyclonal antibodies against FG (1:100).

Techniques: Immunofluorescence, Labeling

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