gal3  (Proteintech)

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: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Spinal cord injury (SCI) increases galectin-3 (GAL3) expression in spinal neurons. (A) Relative GAL3 mRNA expression in the spinal cord after SCI. One-way ANOVA, n = 3/group. (B) Western blot analysis of GAL3 protein after SCI. (C) Statistical data show relative GAL3 protein expression after SCI. One-way ANOVA, n = 3/group. (D) Enzyme-linked immunosorbent assay (ELISA) detection of GAL3 protein levels in rat serum after SCI. One-way ANOVA, n = 6/group. (E) Immunofluorescence microscopy reveals GAL3 co-localization with NeuN post-SCI. (F) Fluorescence intensity of GAL3 after SCI. One-way ANOVA, n = 3/group. (G,H) Immunofluorescence double staining of GAL3 and GFAP (G) or IBA1 (H) after SCI. (I) Determination of optimal glutamate concentration and duration using CCK8 assay. (J) Relative GAL3 mRNA expression in the glutamate-stimulated spinal cord neurons. Unpaired Student’s t -test, n = 3/group. (K) Western blot analysis of GAL3 protein in neuronal injury model. (L) Relative GAL3 protein expression in neuronal injury model. Unpaired Student’s t -test, n = 3/group. (M) ELISA detection of GAL3 in cell supernatant of neuronal injury model. Unpaired Student’s t -test, n = 3/group. (N) Immunofluorescence microscopy showing GAL3 expression in neuronal injury model. (O) Quantification of GAL3 fluorescence intensity in neuronal injury model. Unpaired Student’s t -test, n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Fluorescence, Double Staining, Concentration Assay, CCK-8 Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Galectin-3 (GAL3) contributes to spinal cord injury (SCI)-induced motor impairment. (A) The mRNA level of GAL3 after siR-GAL3 treatment. Unpaired Student’s t -test, n = 3/group. (B) Western blot shows the protein level of GAL3 after siR-GAL3 treatment. (C) Statistical data show the knockdown of GAL3 by siRNA. Unpaired Student’s t -test, n = 3/group. (D) Enzyme-linked immunosorbent assay (ELISA) shows the secretory GAL3 in the supernatant of neurons after siRNA treatment. Unpaired Student’s t -test, n = 3/group. (E) The Basso-Beattie-Bresnahan (BBB) locomotor scores were increased after siR-GAL3 or inhibitor treatment. Two-way Repeated Measures ANOVA, n = 8/group. (F) The inclined plane angles were increased after siR-GAL3 or inhibitor treatment. Two-way Repeated Measures ANOVA, n = 8/group. When SCI + siR-GAL3 group was compared with SCI + Vehicle group, ** P < 0.01, *** P < 0.001; when SCI + TD139 group was compared with SCI + Vehicle group, # P < 0.05, ### P < 0.001; when SCI + GAL3 group was compared with SCI + Vehicle group, + P < 0.05,++ P < 0.01.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Galectin-3 (GAL3) is closely related to programmed cell death after spinal cord injury (SCI). (A) The four datasets before the batch effect were removed. (B) The four datasets after the batch effect were removed. (C) Volcano map shows DEGs in the SCI dataset. (D) Biological Process (BP) analysis of Gene Set Enrichment Analysis (GSEA) in the SCI dataset. Each column represents the P -value score of the pathway between the Sham group and the SCI group, with red indicating upregulation of the pathway in the SCI group, and blue indicating downregulation. (E) Protein-Protein Interaction Networks (PPI) analysis of differentially expressed genes (DEGs) in SCI dataset. In the PPI nodes, red signifies an increase in expression level, while blue indicates a decrease. The intensity of the color corresponds to the magnitude of the differential expression, with darker shades representing a higher differential expression multiple.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Expressing, Quantitative Proteomics

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Galectin-3 (GAL3) regulates neuronal autophagy. (A) Western blot analysis of GAL3 and neuronal autophagy markers ATG7, P62, and LC3 II/I in neurons. (B-E) Quantification of western blot detection of GAL3 (B) , ATG7 (C) , P62 (D) , and LC3 II/I (E) in neurons. One-way ANOVA, n = 3/group. (F) Western blot analysis of GAL3 and neuronal autophagy markers ATG7, P62, and LC3 II/I in the spinal cord of rats. (G–I) Quantification of western blot detection of ATG7 (G) , P62 (H) , and LC3 II/I (I) in the spinal cord of rats. One-way ANOVA, n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Sequencing analysis of spinal cord neurons with galectin-3 (GAL3) knocked down. (A) Volcano map shows differential expression genes (DEGs) in the neuron dataset. (B) Biological process (BP) analysis of Gene Set Enrichment Analysis (GSEA) in the neuron dataset. Each column represents the P -value score of the pathway between the Sham group and the spinal cord injury (SCI) group, with red indicating upregulation of the pathway in the SCI group, and blue indicating downregulation. (C) The Protein-Protein Interaction Networks (PPI) analysis of DEGs in the neuron dataset. In the PPI nodes, red indicates that the expression level increases and blue indicates that the expression level decreases. The darker the color, the greater the differential expression multiple.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Sequencing, Quantitative Proteomics, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Galectin-3 (GAL3) interacts with Cell-division-cycle-42 (CDC42) to regulate neuronal autophagy. (A) Intersected 29 core nodes from the neuron dataset with 22 core nodes from the spinal cord injury (SCI) dataset by the Venn diagram. (B) Correlation analysis between GAL3 and CDC42 expression level in SCI dataset. (C) Correlation analysis between GAL3 and CDC42 expression level in the neuron dataset. (D) Co-immunoprecipitation (Co-IP) shows a direct interaction between GAL3 and CDC42 in the glutamate-induced neuronal damage model. (E) Western blot shows the expression of CDC42, ATG7, P62, and LC3 II/I. (F–I) Quantification of western blot detection of CDC42 (F) , ATG7 (G) , P62 (H) , and LC3 II/I (I) . One-way ANOVA, n = 3/group. (J) Enzyme-linked immunosorbent assay (ELISA) detection of CDC42 in cell supernatant of GAL3-injury model. Unpaired Student’s t -test, n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Galactin-3 regulation of CDC42 promotes neuronal autophagy following spinal cord injury

doi: 10.3389/fncel.2025.1622825

Figure Lengend Snippet: Cell-division-cycle-42 (CDC42) contributes to spinal cord injury (SCI)-induced motor function impairment. (A) The mRNA level after siR-CDC42 treatment. Unpaired Student’s t -test, n = 3/group. (B) Enzyme-linked immunosorbent assay (ELISA) shows the secretory CDC42 in the supernatant of neurons after siRNA treatment. Unpaired Student’s t -test, n = 3/group. (C) The Basso-Beattie-Bresnahan (BBB) locomotor scores were increased after siR-CDC42 and ML141 treatment. Two-way Repeated Measures ANOVA, n = 8/group. (D) The inclined plane angles were increased after siR-CDC42 and ML141 treatment. Two-way Repeated Measures ANOVA, n = 8/group. When SCI + siR-CDC42 group was compared with SCI + Vehicle group, * P < 0.05, ** P < 0.01, *** P < 0.001; when SCI + ML141 group was compared with SCI + Vehicle group, ## P < 0.01, ### P < 0.001. (E,F) Detection of the protein expression level of galectin-3 (GAL3) (E) and CDC42 (F) in serum of healthy volunteers and SCI patients by ELISA. Unpaired Student’s t -test, n = 8/group. *** P < 0.001.

Article Snippet: The primary antibody used targeted the following proteins: NeuN (1:500, mouse IgG; No.26975, Proteintech), GAL3 (1:200, mouse IgG; No. 60207, Proteintech), and CDC42 (1:200, rabbit IgG; No.10155, Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – C ) The C-terminus of HscA is exposed on the surface of phagosomes. ( A ) Immunostaining of phagosomes containing conidia of hscA-myc or hscA L -myc strains isolated from A549 cells. Scale bars, 2 μm. ( B ) Relative signal intensities of the respective emission fluorescence along the lines drawn across the phagosomes shown in ( A ). ( C ) Quantification of exposure distance from a representative experiment. Data represent the mean ± SD; n = 16 individual phagosomes were analyzed. ( D and E ) Biotinylation of host cell proteins by HscA-miniTurboID (HscA-mT). ( D ) A549 cells incubated with conidia of strains hscA-mT or mT - hscA were stained with streptavidin and an antibody against RAB7. ( E ) Phagosomes with positive streptavidin (Strep + ) signal were quantified. The number of independent experiments is indicated at the bottom of the bars. ( F and G ) GAL3 is recruited to damaged phagosomes containing A. fumigatus conidia in A549 cells after 8 hours of infection. ( F ) A549 cells incubated with A. fumigatus conidia were immunostained with indicated antibodies. Scale bars, 5 μm. ( G ) Quantification of GAL3 + phagosomes containing conidia in A549 cells. ( H and I ) Phagosomal SYTOX is released to the nucleus upon damage of the phagosome membrane. ( H ) Representative A549 cells whose lysosomes were loaded with SYTOX-Green, were incubated with A. fumigatus for 16 h. CellMask and CFW were used to stain the cell membrane and A. fumigatus cell wall, respectively. ( I ) Cells with SYTOX signal in the nuclei were quantified. ( J – L ) HscA-dependent phagosomal damage in hMDMs. ( J ) hMDMs incubated with A. fumigatus conidia were immunostained with indicated antibodies. Scale bars, 5 μm. DIC, differential interference contrast. ( K ) GAL3 + phagosomes and ( L ) RAB7 + phagosomes in hMDMs were quantified. Statistics: Error bars represent the mean ± SD. For C and E, p -values were calculated using unpaired two-tailed t test. For G, I, K, and L, p -values are calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Gray dots represent the calculated values from individual microscopic images (G, n = 42–47; I, n = 25–28; K and L, n = 46–54), and colored dots represent the summarized result of individual experiments ( n = 3).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Immunostaining, Isolation, Fluorescence, Incubation, Staining, Infection, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – D ) Recruitment of TSG101 and CHMP3 to phagosomes in hMDMs. ( A ) Detection of TSG101 and GAL3, or ( B ) detection of TSG101 and CHMP3 on phagosomes containing A. fumigatus WT conidia in hMDMs. Regions indicated by white or yellow dashed-line frames are enlarged on the right or bottom, respectively. Channel intensity plots show the fluorescence signal across the yellow lines. ( C and D ) Phagosomes positive for ( C ) TSG101 and ( D ) CHMP3 were quantified. ( E – H ) Recruitment of ESCRT components to phagosomes in A549 cells. (E) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated ESCRT markers. Yellow arrows mark phagosomes positive for both tested markers. DIC, differential interference contrast. ( F – H) Phagosomes positive for ( F ) CHMP3, ( G ) TSG101, and ( H ) ALG2 were quantified. A549 cells or p11-KO cells were incubated with conidia of WT or Δ hscA strains for 4 hours. Intracellular Ca 2+ was subsequently chelated by adding 25 μM BAPTA-AM to the medium, followed by an additional 4 hours of incubation at 37°C. (I) Chelation of Ca 2+ reduces the recruitment of p11 to phagosomes. ( J – L ) Recruitment of ANXA2 and ANXA1 to phagosomes. (J) A549 cells were incubated with A. fumigatus WT conidia and immunostained with antibodies against p11, ANXA2, and ANXA1. Yellow arrows indicate phagosomes positive for both tested markers, while white arrows denote a phagosome positive for ANXA2 but negative for p11. Phagosomes positive for ( K ) ANXA2 and ( L ) ANXA1 were quantified. ( M ) HscA, p11, and Ca 2+ -dependent recruitment of GAL3 to phagosomes. Statistics: Error bars represent the mean ± SD; p -values were determined using unpaired two-tailed t test (C and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments is indicated below each bar.

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Fluorescence, Immunostaining, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – C ) Deletion of fungal HscA or human host p11 gene, or chelation of Ca 2+ , increased phagosome maturation. ( A ) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated phagosomal markers: RAB7 and ANXA2 on the top row, and GAL3 and LAMP1 on the bottom row. Yellow arrows label phagosomes positive for both markers, while white arrows mark phagosomes positive for a single marker. Scale bars, 5 μm. ( B and C ) Phagosomes positive for ( B ) RAB7 and ( C ) LAMP1 were quantified. ( D and E ) Activation of TFEB by p11 deletion or Ca 2+ chelation. ( D ) Immunostaining of A549 cells infected with WT conidia. Dashed-line circles indicate the regions of nuclei. Scale bars, 10 μm. ( E ) Cells with TFEB localized in the nuclei were quantified. Statistics: Error bars represent the mean ± SD; p values were determined using one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments for figures B and C is indicated at the base of each bar. For E, grey dots represent the calculated values from individual microscopic images ( n = 41–68) and colored dots represent summarized results from individual experiments ( n = 3 for BAPTA-AM-treated cells and n = 4 for untreated cells).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Immunostaining, Incubation, Marker, Activation Assay, Infection

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: (A) Presence of Y596/Y αD in HscA/Ssb proteins of human pathogenic fungi. The phylogenic tree was constructed using the alignment of HscA orthologs and Hsp70/Ssa proteins. Branches and names of fungal species are color-coded based on the amino acid residue present in the αD domain: green for Y and yellow for F. Branches corresponding to Hsp70/Ssa are highlighted in pink. ( B and C ) C. albicans and C. glabrata cause damages to phagosomes in hMDMs. (B) hMDMs were incubated with yeast cells of C. albicans (top row) or C. glabrata (bottom row) for 2 hours and immunostained with antibody against GAL3. Scale bars, 5 μm. (C) Phagosomes positive for GAL3 were quantified. CgΔssb1 , the mutant strain of C. glabrata lacking SSB1 . ( D and E ) Increased phagosome damage caused by S. cerevisiae expressing Ssb with an L-to-Y mutation. (D) hMDMs were incubated with yeast cells of Ssb Y protein producing S. cerevisiae ( Sc-SSB Y ) or the Ssb L protein producing S. cerevisiae ( Sc-SSB L ) for 2 hours and immunostained with antibodies against GAL3 and RAB7. Yeast cell wall was stained with CFW. Arrows indicate phagosomes positive for both markers. Regions indicated by dashed-line frames are enlarged on the right. Channel intensity plots show the fluorescence signal across the yellow lines. Scale bars, 5 μm. (E) The yeast cells containing phagosomes that are positive for GAL3 were quantified. Sc , S. cerevisiae WT strain; Sc-AD , S. cerevisiae strain transformed with control vector; Sc-AfHscA , S. cerevisiae strain producing A. fumigatus HscA; Sc-SSB F , S. cerevisiae strain producing Ssb F , Sc-AGA2 , S. cerevisiae strain producing Aga2p. Statistics: Error bars represent the mean ± SD of pooled calculated values, indicated by grey dots, from individual microscopic images ( n = 60 for C. albicans and C. glabrata ; n = 59 for CgΔssb1 ; n = 75–81 for strains of S. cerevisiae ). P- values were calculated using an unpaired two-tailed t test ( C ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( E ). Colored dots represent summarized results from individual experiments ( n = 3 for C and n = 4 for E).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Construct, Residue, Incubation, Mutagenesis, Expressing, Staining, Fluorescence, Transformation Assay, Control, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) UMAP plot showing integrated clustering of immune cells samples from four mouse models of retinal degeneration, including LD model (sorted by Cx3cr1 + ), NaIO 3 model (CD45 + ), P23H model (CD45 + ) and aging model (CD45 + ) and naïve mice (CD45 + ). A total of 15,623 macrophages, including 13,489 microglia, were integrated among four models. PMN, polymorphonuclear neutrophils; mo-MFs, monocyte-derived macrophages; pv-MFs: perivascular macrophages; NK, natural killer. ( B ) UMAP plots showing integrated macrophage clusters by two datasets. Dash circles indicate subretinal microglia (srMG). ( C ) Percentage of sample distribution by clusters. The arrow indicates the enrichment of srMG cluster from degenerating retinas. ( D ) Heatmap of top 30 conserved marker genes of subretinal microglia shared by each model across clusters. Genes were ranked by fold changes. Arrows indicate srMG cluster. ( E ) In situ validation of Gal3 expression on the apical RPE (top) or in the neuroretina from the inner plexiform layer (bottom). Iba1 (green), phalloidin (red, only in RPE) and Gal3 (magenta). Scale bar: 100μm. ( F ) Percentage of Gal3 + cells relative to Iba1 + cells between RPE and neuroretina tissues across models.

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Derivative Assay, Marker, In Situ, Expressing

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) Iba1 (green) and phalloidin (red) staining in RPE flatmounts from LD-subjected mice as indicated. ( B ) Quantifications of subretinal Iba1 + cells as shown in A. ( C ) Iba1 (green) and phalloidin (red) staining in RPE flatmounts from P23H mice as indicated. ( D ) Quantifications of subretinal Iba1 + cells as shown in C. ( E ) Examples of ERG responses at different flash intensities as indicated. ( F ) Representative retinal cross sections of WT, Lgal3 +/- and Lgal3 -/- in P23H mice. ( G and H ) Quantifications of Gal3 depletion efficiency (G) and frequencies of subretinal Iba1 + cells (H) in Gal3 cKO mice (n=9) compared with genotype control mice (n=9) and tamoxifen control (n=8). Scale bars: 100 μm. Data were collected from 2-3 independent experiments. ***: p<0.001; ns: not significant (one-way ANOVA with Tukey’s post hoc test).

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Staining

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) Images of phalloidin staining in WT and Lgal3 -/- RPE tissues in LD. ( B ) Quantifications of dysmorphic RPE cells (n=6, 7 and 3, respectively). ( C ) TUNEL (green) and DAPI (blue) staining in WT and Lgal3 -/- retinal cross sections in LD. ONL and INL, outer and inner nuclear layers. ( D ) Quantifications of TUNEL + photoreceptors in ONL (n=5, 5 and 3, respectively). ( E ) Rhodopsin (red) and Iba1 (green) staining in WT and Lgal3 -/- retinal cross sections in LD. Images from single planes of confocal scans were shown. ( F ) Quantifications of rhodopsin+ subretinal microglia (n=4 per group). ( G ) Images of phalloidin staining in WT and Lgal3 -/- RPE tissues at 2 years of age. ( H ) Quantifications of RPE cell size. Dots represent individual images with n=5 mice per group. ( I ) ERG data showing scotopic a- and b-waves in 2-year-old WT (n=5) and Lgal3 -/- (n=5) mice. ( J ) Scotopic a- and b-waves of ERG data among Lgal3 +/+ (n=12), Lgal3 +/- (n=6) and Lgal3 -/- (n=10) in P23H mice. ( K ) Quantifications of ONL thickness among Lgal3 +/+ (n=7), Lgal3 +/- (n=7), and Lgal3 -/- (n=8) in P23H mice. ( L ) Representative images of dysmorphic RPE cells in Gal3 cKO in LD. Iba1, green; phalloidin, red; Gal3, magenta. ( M ) Quantifications of dysmorphic RPE cells in Gal3 cKO mice (n=9) compared with genotype control ( Cx3cr1 CreER/+ Lgals3 fl/fl mice, n=9) and tamoxifen control ( Cx3cr1 CreER/+ mice treated with tamoxifen, n=8). Scale bars: 100μm. Data were collected from 2-3 independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. One-way ANOVA with Tukey’s post hoc test (B, D and M); unpaired Student’s t-test (F and H); two-way ANOVA with Tukey’s post hoc test (I, J and K).

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Staining, TUNEL Assay

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) Violin plots showing the upregulation of genes ( Lgals3, Syk and Ctnnb1I) related to Trem2 signaling by subretinal microglia from the integrated dataset of all four mouse models. ( B ) Images of Iba1 (green) and Trem2 (red) staining in naïve microglia from inner retina and subretinal microglia in LD. ( C ) Images of Iba1 (green) and Syk (red) staining in subretinal microglia and microglia from inner retina in LD. ( D ) 3D rendering images of Gal3 (green), Trem2 (red) and Iba1 (white) staining in subretinal microglia in LD. Views from both the apical RPE aspect and neuroretina aspect are shown. ( E ) Images of Iba1 (green), Trem2 (red) and Gal3 (magenta) staining in subretinal microglia between control and Trem2 cKO mice in LD. ( F-H ) Quantifications of Trem2 depletion (F, n=4 per group), Iba1 + cells (G, n=9) and Gal3 + cells (H, n=9) between control and Trem2 cKO mice. ( I ) Fundus images showing increased subretinal white lesions in of Trem2 cKO mice in LD as indicated by arrows. Images from four individual mice per group are shown. ( J ) Images of phalloidin staining in RPE tissues from control and Trem2 cKO mice in LD. ( K ) Quantifications of dysmorphic RPE cells between control and Trem2 cKO mice (n=9 per group). Scale bars: 50μm (D); 100μm (B, C E,and J). Data were collected from 2 independent experiments. **: p<0.01; ***: p<0.001. Unpaired Student’s t-test (F-H).

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Staining

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) Split views of confocal scans showing the colocalization of Trem2 (red) and Gal3 (green) in the subretinal microglia. Lines indicate the RPE-facing and neuroretina (NR)-facing aspects as indicated. ( B ) Fundus images showing increased subretinal white lesions in anti-Trem2 mAb178 treated mice in LD as indicated by arrows. Images of 4 individual mice per group are shown. ( C ) Images of Iba1 (green) and Gal3 (magenta) staining in subretinal microglia between control and mAb178-treated mice in LD. Scale bar: 100 μm. ( D and E ) Quantifications of Iba1 + cells and Gal3 + cells between control and mAb178 (n=8 per group). ( F ) Images of phalloidin staining in RPE flatmounts from control and mAb178 treated mice in LD. Scale bar: 100μm. ( G ) Quantifications of dysmorphic RPE cells between control (n=8) and mAb178 (n=9) treated mice. ( H ) Images of Iba1 (green) and Trem2 (red) in microglia from the inner retina of naïve control and Trem2 cKO mice. Scale bar: 50μm.

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Staining

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) ELISA of soluble Trem2 (sTrem2) in vitreous fluid and retinal fluid from naïve WT mice, WT and Trem2 cKO mice subjected to LD. ( B ) Fundus images of mice treated with isotype control or 4D9 anti-Trem2 in LD. Four individual mice per group are shown. ( C ) Representative OCT images of mice treated with isotype or 4D9 in LD. ( D ) Quantifications of outer nuclear layer (ONL) thickness by OCT (n=13 per group). ONL thickness was measured at both nasal and temporal sides. ( E and F ) Scotopic a-waves and b-waves of ERG data among mice treated with isotype or 4D9 in naïve or LD setting (n=5 per group). ( G ) Fundus images of Gal3 cKO mice treated with isotype or 4D9 in LD. Four individual mice per group are shown. ( H ) Representative OCT images of Gal3 cKO mice treated with isotype control or 4D9 anti-Trem2 in LD. ( I ) Quantifications of average ONL thickness by OCT between control and Gal3 cKO mice treated with either isotype or 4D9 (n=13 per group). ( J ) Images of phalloidin staining of control and Gal3 cKO RPE treated with isotype or 4D9 in LD. ( K ) Quantifications of dysmorphic RPE cells (n=15, 13, 11 and 13, respectively). Scale bars: 100μm. Data were collected from 2-4 independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. Unpaired Student’s t-test (F-H). One-way ANOVA with Tukey’s post hoc test (A); two-way ANOVA with Tukey’s post hoc test (D-F, I and K).

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) Staining of human IgG (red) and Iba1 (green) in retinal cross sections collected from mice with or without 4D9 treatment in LD. The hIgG is used to trace 4D9 antibodies, which outlines retinal vasculatures in 4D9 treated mice. Arrows indicate the presence of 4D9 antibodies in the subretinal microglia, while asters indicate the absence of 4D9 antibodies in microglia from the inner retina. ( B ) Human IgG (red) and Iba1 (green) staining in RPE and neuroretina flatmounts collected from mice treated with 4D9 antibodies in LD. ( C ) Quantifications of hIgG + microglia in the subretinal space and neuroretina. ( D and E ) Quantifications of Iba1 + cells and Gal3 + cells between control and Gal3 cKO mice treated with either isotype or 4D9 (n=13 per group). Scale bars: 100 μm. Data were collected from 2-4 independent experiments. ***: p<0.001; ns: not significant (unpaired Student’s t-test: C; two-way ANOVA with Tukey’s post hoc test: D and E).

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Staining

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) Multispectral imaging of GAL3 and CD68 co-staining in the subretinal space (top) and inner retina (bottom) from human donors. Unmixed purple spectrum (GAL3) and yellow spectrum (CD68) are shown. The areas of colocalized spectra are highlighted in green. Scale bar: 50μm. ONL and INL, outer and inner nuclear layers. ( B ) Representative image of Gal3 and CD68 co-staining in the macular GA region of a retinal section from an 88-year-old female donor eye with advanced AMD (Sarks V). Black insert box shows the magnification of GA with double positive cells. Scale bar: 200μm. ONL and INL, outer and inner nuclear layers; GCL, ganglion cell layer. ( C ) Correlation between the frequencies of macular Gal3 + CD68 + double positive cells (y axis) and Sarks AMD grading (x axis) by Spearman’s correlation (n = 18 donors, Table S2). Coefficient and p-value are shown. ( D ) Histograms showing increased TREM2 + myeloid cells (CD45 + CD11B + ) in RPE/choroid tissues of AMD donors. Concatenated histograms were shown (n=3 per groups). Control human blood samples were used to set up flow gating. ( E-G ) Quantifications of TREM2 + (E), CD45 + (F), and CD11B + (G) cell frequencies in RPE/choroid tissues between non-AMD and AMD donors. Unpaired Student’s t test is used. P-values are shown. ( H ) Correlation between the frequencies of TREM2 + myeloid cells (y axis) and Sarks AMD grading (x axis) in RPE/choroid tissues by Spearman’s correlation. Coefficient and p-value are shown.

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Imaging, Staining

Journal: bioRxiv

Article Title: Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions

doi: 10.1101/2023.07.19.549403

Figure Lengend Snippet: ( A ) Images of GAL3 (purple) and CD68 (yellow) co-staining in the macula region of retinal sections from human donors categorized by Sark grades (I-VI). The macular neurosensory retinas of some subject eyes exhibited fixation-related artifactual detachment. In these subjects, separate images of RPE/choroid tissues are shown. Scale bar: 100μm. ONL and INL, outer and inner nuclear layers. GCL, ganglion cell layer. ( B ) Spectral imaging of GAL3 and CD68 co-staining in the geographic atrophy from donor #23 with advanced AMD (Sarks V). Unmixed purple spectrum (GAL3) and yellow spectrum (CD68) are shown. The areas of colocalized spectra are highlighted in green. Scale bar: 50μm. ( C and D ) Images showing the presence of subretinal GAL3 (purple) and CD68 (yellow) double positive cells in the areas with photoreceptor loss and preserved RPE in the transitional area of the macula from an AMD donor (C) and in the age-related peripheral degeneration of a non-AMD donor (D). Scale bars: 100μm. ( E ) Gating strategy of flow cytometry analysis. CD45 + CD11B + cells and CD45 + CD11B - cells from control blood were used to determine the gating of TREM2 + cells. Concatenated plots are shown for non-AMD and AMD. ( F ) Flow contour plots of individual donors showing increased percentage of TREM2 + myeloid cells in AMD.

Article Snippet: Primary antibodies used were as follows: rabbit anti-Iba1 (Wako #019-19741), goat anti-Gal3 (R&D #AF1197), rat anti-Gal3 (Biolegend #125401), sheep anti-Trem2 (R&D #AF1729), mouse anti-rhodopsin (Abcam #ab5417) and rabbit anti-Syk (Abcam #ab40781).

Techniques: Staining, Imaging, Flow Cytometry