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ab134047 abcam rabbit antirat igg 3 33 ai 4001 vector laboratories ly6g 3 33 nbp2 00441 novus biologicals cd68 2 5 (Vector Laboratories)

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Vector Laboratories ab134047 abcam rabbit antirat igg 3 33 ai 4001 vector laboratories ly6g 3 33 nbp2 00441 novus biologicals cd68 2 5
Ab134047 Abcam Rabbit Antirat Igg 3 33 Ai 4001 Vector Laboratories Ly6g 3 33 Nbp2 00441 Novus Biologicals Cd68 2 5, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( A ) UMAP plot of microglia separated into transcriptional clusters (n=1 pool of five animals for each age). ( B ) Superimposition of ages onto the UMAP plot with dashed lines signifying relative cluster demarcation. ( C ) Percent composition of each cluster by age. ( D ) Dotplot of expression of cluster markers sorted by age. Percent of cells expressing the gene and average normalized expression are represented. ( E ) Violin plots of genes with dynamic age-related expression patterns. ( F ) Representative images and quantification of C1q (yellow), C3 (red), and Iba1 (cyan) staining in the hilus and molecular layer (ML) of 6- and 24-month-old mice (n=4–5 mice per group; mixed effects analysis followed by Dunnett’s multiple comparisons; *p<0.05, **p<0.01, ****p<0.0001). ( G ) Number of differentially expressed genes from the 6-month timepoint at each age. Bars above the intersect represent increased expression and those below represent decreased expression. ( H ) The average expression change at all ages for genes differentially regulated genes at individual ages represented by the color scheme in ( B, I, J, K ), Volcano plots of differentially expressed genes in microglia between 6 and 12 months ( I ), 6 and 18 months ( J ), and 6 and 24 months ( K ) and corresponding gene ontology analysis of genes with significantly increased (brown) or decreased (green) expression for each comparison. Data are shown as mean ± s.e.m.

Journal: eLife

Article Title: Microglia aging in the hippocampus advances through intermediate states that drive activation and cognitive decline

doi: 10.7554/eLife.97671

Figure Lengend Snippet: ( A ) UMAP plot of microglia separated into transcriptional clusters (n=1 pool of five animals for each age). ( B ) Superimposition of ages onto the UMAP plot with dashed lines signifying relative cluster demarcation. ( C ) Percent composition of each cluster by age. ( D ) Dotplot of expression of cluster markers sorted by age. Percent of cells expressing the gene and average normalized expression are represented. ( E ) Violin plots of genes with dynamic age-related expression patterns. ( F ) Representative images and quantification of C1q (yellow), C3 (red), and Iba1 (cyan) staining in the hilus and molecular layer (ML) of 6- and 24-month-old mice (n=4–5 mice per group; mixed effects analysis followed by Dunnett’s multiple comparisons; *p<0.05, **p<0.01, ****p<0.0001). ( G ) Number of differentially expressed genes from the 6-month timepoint at each age. Bars above the intersect represent increased expression and those below represent decreased expression. ( H ) The average expression change at all ages for genes differentially regulated genes at individual ages represented by the color scheme in ( B, I, J, K ), Volcano plots of differentially expressed genes in microglia between 6 and 12 months ( I ), 6 and 18 months ( J ), and 6 and 24 months ( K ) and corresponding gene ontology analysis of genes with significantly increased (brown) or decreased (green) expression for each comparison. Data are shown as mean ± s.e.m.

Article Snippet: The following primary antibodies were used: rabbit anti-IBA1 (1:1000, Wako, Cat# 019-19741, RRID: AB_839504 ), rat anti-CD68 (1:250, Bio-Rad, Cat# MCA1957, RRID: AB_322219 ), rabbit anti-NFKB p65 (1:500, Santa Cruz, Cat# sc-372, RRID: AB_632037 ), guinea pig anti-Iba1 (1:1000, Synaptic Systems, Cat# 234-004, RRID: AB_2493179 ), rabbit anti-C1q (1:1000, Abcam, Cat# ab182451, RRID: AB_2732849 ), rat anti-C3 (1:1000, Abcam, Cat# ab11862, RRID: AB_2066623 ), rabbit anti-S6 (1:500, Cell Signaling, Cat# 2217, RRID: AB_331355 ), and rabbit anti-KLF2 (1:250, Bioss, Cat# bs-2772R, RRID: AB_10857057 ).

Techniques: Expressing, Staining, Comparison

( A ) Diagram depicting ages utilized for immunohistochemical analysis. ( B ) Diagram of the hippocampus labeled with the regions analyzed. ( C ) Illustration of subregions analyzed by immunohistochemistry. ( D ) Representative images and quantification of IBA1 (cyan)/CD68 (red)-positive microglia across hippocampal subregions in 3- and 24-month-old mice. Scale bars are 10 μM (n=5 per group; t -test with Holm–Sidak correction; *p<0.05, **p<0.01). ( E ) Heatmap of the quantification of activated microglia across ages and subregions. ( F ) Representative wide-field images of IBA1 (cyan)/CD68 (red) in the dentate gyrus in 3- and 24-month-old mice. Scale bars are 100 μM. ( G ) Representative wide field images of Iba1 (cyan)/NFKB p65 (yellow) in the dentate gyrus in 3- and 24-month-old mice. Scale bars are 100 μM. ( H ) Representative images and quantification of Iba1 (cyan)/NFKB p65 (yellow) staining across hippocampal subregions. Scale bars are 10 μM (n=5 per group; t -test with Holm–Sidak correction; *p<0.05, **p<0.01, ****p<0.0001). ( I ) Heatmap of the quantification of NFKB signal in microglia across ages and subregions. n=4–5 mice per condition.

Journal: eLife

Article Title: Microglia aging in the hippocampus advances through intermediate states that drive activation and cognitive decline

doi: 10.7554/eLife.97671

Figure Lengend Snippet: ( A ) Diagram depicting ages utilized for immunohistochemical analysis. ( B ) Diagram of the hippocampus labeled with the regions analyzed. ( C ) Illustration of subregions analyzed by immunohistochemistry. ( D ) Representative images and quantification of IBA1 (cyan)/CD68 (red)-positive microglia across hippocampal subregions in 3- and 24-month-old mice. Scale bars are 10 μM (n=5 per group; t -test with Holm–Sidak correction; *p<0.05, **p<0.01). ( E ) Heatmap of the quantification of activated microglia across ages and subregions. ( F ) Representative wide-field images of IBA1 (cyan)/CD68 (red) in the dentate gyrus in 3- and 24-month-old mice. Scale bars are 100 μM. ( G ) Representative wide field images of Iba1 (cyan)/NFKB p65 (yellow) in the dentate gyrus in 3- and 24-month-old mice. Scale bars are 100 μM. ( H ) Representative images and quantification of Iba1 (cyan)/NFKB p65 (yellow) staining across hippocampal subregions. Scale bars are 10 μM (n=5 per group; t -test with Holm–Sidak correction; *p<0.05, **p<0.01, ****p<0.0001). ( I ) Heatmap of the quantification of NFKB signal in microglia across ages and subregions. n=4–5 mice per condition.

Techniques: Immunohistochemical staining, Labeling, Immunohistochemistry, Staining

( A ) Pseudotime trajectories of microglia from an anchor point located in 6-month microglia presented in a UMAP plot (n=1 pool of five animals for each age). ( B ) Microglia ages superimposed over pseudotime trajectories. ( C ) Gene expression modules representing sections of the inflammatory aging trajectory over the right half of the UMAP plot (left). Modules were discovered using Moran’s I autocorrelation test. Top gene ontology terms and representative genes in each module (right). ( D ) Dotplot of pseudotime modules sorted by age. Percent of cells expressing the gene and average normalized expression are represented. ( E–G ) Average gene expression changes for each aging module represented as log2 fold change of 12 months ( E ), 18 months ( F ), or 24 months ( G ) over 6 months. ( H ) Representative images and quantification of KLF2 (magenta) and IBA1 (cyan) staining in the hippocampus across ages (n=3 mice per group; one-way ANOVA with Tukey’s post-hoc test; *p<0.05). ( I ) Diagram of the heterochronic parabiosis model with the comparisons made in scRNA-Seq. ( J ) Representative images and quantification of CD68 (red) and IBA1 (cyan) staining in the hippocampus of isochronic young (IY) and heterochronic young (HY) (n=5 mice per group; unpaired Student’s t -test; ***p<0.001). ( K ) Average gene expression changes for each aging module represented as log2 fold change of heterochronic young (HY) over isochronic young (IY) adult parabionts. Data from . ( L ) Diagram of microglia surrounding an Aβ plaque. ( M ) Average gene expression changes for each aging module represented as log2 fold change of the App NL-G-F genotype (AD) over wildtype (WT). Data from (one-sample t -test with the expected value of 0 [no change]; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Data are shown as mean ± s.e.m.

Journal: eLife

Article Title: Microglia aging in the hippocampus advances through intermediate states that drive activation and cognitive decline

doi: 10.7554/eLife.97671

Figure Lengend Snippet: ( A ) Pseudotime trajectories of microglia from an anchor point located in 6-month microglia presented in a UMAP plot (n=1 pool of five animals for each age). ( B ) Microglia ages superimposed over pseudotime trajectories. ( C ) Gene expression modules representing sections of the inflammatory aging trajectory over the right half of the UMAP plot (left). Modules were discovered using Moran’s I autocorrelation test. Top gene ontology terms and representative genes in each module (right). ( D ) Dotplot of pseudotime modules sorted by age. Percent of cells expressing the gene and average normalized expression are represented. ( E–G ) Average gene expression changes for each aging module represented as log2 fold change of 12 months ( E ), 18 months ( F ), or 24 months ( G ) over 6 months. ( H ) Representative images and quantification of KLF2 (magenta) and IBA1 (cyan) staining in the hippocampus across ages (n=3 mice per group; one-way ANOVA with Tukey’s post-hoc test; *p<0.05). ( I ) Diagram of the heterochronic parabiosis model with the comparisons made in scRNA-Seq. ( J ) Representative images and quantification of CD68 (red) and IBA1 (cyan) staining in the hippocampus of isochronic young (IY) and heterochronic young (HY) (n=5 mice per group; unpaired Student’s t -test; ***p<0.001). ( K ) Average gene expression changes for each aging module represented as log2 fold change of heterochronic young (HY) over isochronic young (IY) adult parabionts. Data from . ( L ) Diagram of microglia surrounding an Aβ plaque. ( M ) Average gene expression changes for each aging module represented as log2 fold change of the App NL-G-F genotype (AD) over wildtype (WT). Data from (one-sample t -test with the expected value of 0 [no change]; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Data are shown as mean ± s.e.m.

Techniques: Gene Expression, Expressing, Staining

( A ) Volcano plot of differential gene expression of 12-month microglia versus all other ages with significant genes in teal. ( B ) Gene ontology analysis of biological processes enriched in those genes with increased expression at 12 months of age. ( C ) Representative images and quantification of CD68 (red) and IBA1 (cyan) staining in the hippocampus of isochronic young (IY) and heterochronic young (HY) along with a diagram of the comparisons (n=5 mice per group; unpaired Student’s t -test; ***p<0.001). Data are shown as mean ± s.e.m. ( D ) Dotplot of pseudotime modules in young (Y), isochronic young (IY), and heterochronic young (HY) parabiont microglia. Data is from . Percent of cells expressing the gene and average normalized expression are represented. ( E ) Volcano plots of differential gene expression of App NL-G-F genotype (AD) over wildtype (WT) microglia at 6 (left) and 12 months of age. Significant genes are in teal. Data is from . ( F ) Dotplot of pseudotime modules across ages (3, 6, 12, and 21 months old) and genotypes ( App NL-G-F genotype (AD) over C57Bl/6 (WT)). Data is from . (G) Percent of cells expressing the gene and average normalized expression are represented.

Journal: eLife

Article Title: Microglia aging in the hippocampus advances through intermediate states that drive activation and cognitive decline

doi: 10.7554/eLife.97671

Figure Lengend Snippet: ( A ) Volcano plot of differential gene expression of 12-month microglia versus all other ages with significant genes in teal. ( B ) Gene ontology analysis of biological processes enriched in those genes with increased expression at 12 months of age. ( C ) Representative images and quantification of CD68 (red) and IBA1 (cyan) staining in the hippocampus of isochronic young (IY) and heterochronic young (HY) along with a diagram of the comparisons (n=5 mice per group; unpaired Student’s t -test; ***p<0.001). Data are shown as mean ± s.e.m. ( D ) Dotplot of pseudotime modules in young (Y), isochronic young (IY), and heterochronic young (HY) parabiont microglia. Data is from . Percent of cells expressing the gene and average normalized expression are represented. ( E ) Volcano plots of differential gene expression of App NL-G-F genotype (AD) over wildtype (WT) microglia at 6 (left) and 12 months of age. Significant genes are in teal. Data is from . ( F ) Dotplot of pseudotime modules across ages (3, 6, 12, and 21 months old) and genotypes ( App NL-G-F genotype (AD) over C57Bl/6 (WT)). Data is from . (G) Percent of cells expressing the gene and average normalized expression are represented.

Techniques: Gene Expression, Expressing, Staining

( A ) Representation of the microglia aging trajectory over the UMAP plot highlighting the region of peak Tgfb1 expression. ( B ) Representative RNAscope images and quantification of Tgfb1 (red) expression in IBA1 (cyan) cells across ages (n=5 per group; one-way ANOVA with Dunnett’s post hoc test; *p<0.05). ( C ) Dotplot of the expression values of TGFB1 signaling components from scRNA-Seq of aging hippocampal microglia (6-, 12-, 18-, and 24-month-old). Percent of cells expressing the gene and average normalized expression are represented. ( D ) Schematic of the heterochronic parabiosis model and quantification of hippocampal microglia expression of Tgfb1 from isochronic young (IY) and heterochronic young (HY) adult parabionts. Data derived from . ( E ) Top gene ontology terms for the set of genes with significantly decreased expression in bulk microglia RNA-Seq following TGFB1 treatment compared to control (DMSO) in LPS-treated microglia (n=5 per group). ( F ) Heatmap of top 10 genes in each aging module following TGFB1 compared to DMSO in LPS-treated microglia. ( G ) Average gene expression changes for each aging module represented as log2 fold change of TGFB1 treatment over DMSO (one-sample t -test with the expected value of 0 [no change]; *p<0.05, ***p<0.001, ****p<0.0001). ( H ) Representation of the microglia aging trajectory over the UMAP plot highlighting the stage where CX-5461 modulates the trajectory. ( I ) Representative images of S6 (magenta) and Iba1 (cyan) staining in the hippocampus of 6- and 24-month-old mice and quantification across aging (n=3 mice per group; one-way ANOVA with Tukey’s post hoc test; *p<0.05, **p<0.005, ****p<0.0001). ( J ) Schematic of the heterochronic parabiosis model and quantification of hippocampal microglia expression of translation module from isochronic young (IY) and heterochronic young (HY) adult parabionts. Data derived from (one-sample t -test with the expected value of 0 [no change]; **p<0.01). ( K ) Top gene ontology terms for the set of genes with significantly decreased expression in bulk microglia RNA-Seq following CX-5461 treatment compared to control (DMSO) in LPS-treated microglia (n=3 per group) ( L ) Heatmap of top 10 genes in each aging module following CX-5461 compared to DMSO in LPS-treated microglia. ( M ) Average gene expression changes for each aging module represented as log2 fold change of CX-5461 treatment over DMSO (one-sample t -test with the expected value of 0 [no change]; *p<0.05, **p<0.01, ****p<0.0001).

Journal: eLife

Article Title: Microglia aging in the hippocampus advances through intermediate states that drive activation and cognitive decline

doi: 10.7554/eLife.97671

Figure Lengend Snippet: ( A ) Representation of the microglia aging trajectory over the UMAP plot highlighting the region of peak Tgfb1 expression. ( B ) Representative RNAscope images and quantification of Tgfb1 (red) expression in IBA1 (cyan) cells across ages (n=5 per group; one-way ANOVA with Dunnett’s post hoc test; *p<0.05). ( C ) Dotplot of the expression values of TGFB1 signaling components from scRNA-Seq of aging hippocampal microglia (6-, 12-, 18-, and 24-month-old). Percent of cells expressing the gene and average normalized expression are represented. ( D ) Schematic of the heterochronic parabiosis model and quantification of hippocampal microglia expression of Tgfb1 from isochronic young (IY) and heterochronic young (HY) adult parabionts. Data derived from . ( E ) Top gene ontology terms for the set of genes with significantly decreased expression in bulk microglia RNA-Seq following TGFB1 treatment compared to control (DMSO) in LPS-treated microglia (n=5 per group). ( F ) Heatmap of top 10 genes in each aging module following TGFB1 compared to DMSO in LPS-treated microglia. ( G ) Average gene expression changes for each aging module represented as log2 fold change of TGFB1 treatment over DMSO (one-sample t -test with the expected value of 0 [no change]; *p<0.05, ***p<0.001, ****p<0.0001). ( H ) Representation of the microglia aging trajectory over the UMAP plot highlighting the stage where CX-5461 modulates the trajectory. ( I ) Representative images of S6 (magenta) and Iba1 (cyan) staining in the hippocampus of 6- and 24-month-old mice and quantification across aging (n=3 mice per group; one-way ANOVA with Tukey’s post hoc test; *p<0.05, **p<0.005, ****p<0.0001). ( J ) Schematic of the heterochronic parabiosis model and quantification of hippocampal microglia expression of translation module from isochronic young (IY) and heterochronic young (HY) adult parabionts. Data derived from (one-sample t -test with the expected value of 0 [no change]; **p<0.01). ( K ) Top gene ontology terms for the set of genes with significantly decreased expression in bulk microglia RNA-Seq following CX-5461 treatment compared to control (DMSO) in LPS-treated microglia (n=3 per group) ( L ) Heatmap of top 10 genes in each aging module following CX-5461 compared to DMSO in LPS-treated microglia. ( M ) Average gene expression changes for each aging module represented as log2 fold change of CX-5461 treatment over DMSO (one-sample t -test with the expected value of 0 [no change]; *p<0.05, **p<0.01, ****p<0.0001).

Techniques: Expressing, RNAscope, Derivative Assay, RNA Sequencing, Control, Gene Expression, Staining

( A ) Quantification of percentage of Tgfb1 RNAscope signal within IBA1 cells. ( B ) Quantification of IHC of pTGFBR1 signal in IBA1 cells across ages (n=4–5 mice per group; one-way ANOVA; *p<0.05). ( C ) Quantification of the expression of TGFB1 signaling components from . Expression values for the genes are represented as Z-scores (n=12 genes with reads in >25% of cells; Friedman test followed by Dunn’s multiple comparisons test; *p<0.05, ***p<0.001). ( D ) Dotplot of the expression values of TGFB1 signaling components in young (Y), aged (A), isochronic young (IY), and heterochronic young (HY) parabiont microglia. Data is from . Percent of cells expressing the gene and average normalized expression are represented. UMAP plot of Tgfb1 scRNA-Seq colored by genotype. ( E ) Dotplot of the expression values of TGFB1 signaling components in aging microglia categorized by the expression of Tgfb1 into quartiles with Q1 having the lowest expression of Tgfb1 and Q4 having the highest expression. ( F ) Quantification of the expression of TGFB1 signaling components from ( E ). Expression values for the genes are represented as Z-scores (n=12 genes with reads in >25% of cells; Friedman test followed by Dunn’s multiple comparisons test; *p<0.05, ***p<0.001). ( G ) Dotplot of the expression values of selected TGFB signaling targets (activated and repressed, genes found in both and ) in aging microglia categorized by the expression of Tgfb1 . ( H ) Heatmap of gene expression changes in the top 10 genes in each aging module induced by LPS. ( I ) Overlap between microglia gene expression changes induced by LPS and aging (χ 2 <2.2e-16). ( J ) PCA plot of pharmacological manipulations with or without LPS treatment.

Journal: eLife

Article Title: Microglia aging in the hippocampus advances through intermediate states that drive activation and cognitive decline

doi: 10.7554/eLife.97671

Figure Lengend Snippet: ( A ) Quantification of percentage of Tgfb1 RNAscope signal within IBA1 cells. ( B ) Quantification of IHC of pTGFBR1 signal in IBA1 cells across ages (n=4–5 mice per group; one-way ANOVA; *p<0.05). ( C ) Quantification of the expression of TGFB1 signaling components from . Expression values for the genes are represented as Z-scores (n=12 genes with reads in >25% of cells; Friedman test followed by Dunn’s multiple comparisons test; *p<0.05, ***p<0.001). ( D ) Dotplot of the expression values of TGFB1 signaling components in young (Y), aged (A), isochronic young (IY), and heterochronic young (HY) parabiont microglia. Data is from . Percent of cells expressing the gene and average normalized expression are represented. UMAP plot of Tgfb1 scRNA-Seq colored by genotype. ( E ) Dotplot of the expression values of TGFB1 signaling components in aging microglia categorized by the expression of Tgfb1 into quartiles with Q1 having the lowest expression of Tgfb1 and Q4 having the highest expression. ( F ) Quantification of the expression of TGFB1 signaling components from ( E ). Expression values for the genes are represented as Z-scores (n=12 genes with reads in >25% of cells; Friedman test followed by Dunn’s multiple comparisons test; *p<0.05, ***p<0.001). ( G ) Dotplot of the expression values of selected TGFB signaling targets (activated and repressed, genes found in both and ) in aging microglia categorized by the expression of Tgfb1 . ( H ) Heatmap of gene expression changes in the top 10 genes in each aging module induced by LPS. ( I ) Overlap between microglia gene expression changes induced by LPS and aging (χ 2 <2.2e-16). ( J ) PCA plot of pharmacological manipulations with or without LPS treatment.

Techniques: RNAscope, Expressing, Gene Expression

( A ) Representative images and quantification of IBA1 (cyan)/CD68 (red)-positive microglia in wildtype, Tgfb1 cHet, and Tgfb1 cKO hippocampi (n=3–5; one way ANOVA with Tukey post hoc test; *p<0.05). ( B ) Dotplot of the expression values of TGFB1 signaling components from scRNA-Seq of Tgfb1 WT, cHet, and cKO microglia (n=2 pools of three animals per genotype). ( C ) UMAP plot of Tgfb1 scRNA-Seq colored by genotype. ( D ) FACS plots of gating strategy for microglia isolation for RNA-Seq in cHet and cKO hippocampi. Quantification of the flow cytometry analysis of CD11b and CD45 (n=3–5; mixed effects analysis; *p<0.05, ****p<0.0001). ( E ) Example histogram and quantification of CD48 in control and Tgfb1 cKO FACS-sorted microglia (n=3–5 per group; t -test; ****p<0.0001). ( F ) Top gene ontology terms associated with genes significantly increased in microglia bulk RNA-Seq in Tgfb1 cKO samples (n=3–5 samples per genotype). ( G ) Top gene ontology terms associated with genes significantly decreased in microglia bulk RNA-Seq in Tgfb1 cKO samples. ( H ) Overlap between concordant microglia gene expression changes induced by Tgfb1 knockout and aging. (χ 2 <2.2e-16). ( I ) Heatmap of gene expression of the top 10 genes in each aging module in control and Tgfb1 cKO microglia. ( J ) Average gene expression changes for each aging module represented as log2 fold change of Tgfb1 cKO over control (one-sample t -test with the expected value of 0 [no change]; ****p<0.0001). Data are shown as means ± s.e.m.

Journal: eLife

Article Title: Microglia aging in the hippocampus advances through intermediate states that drive activation and cognitive decline

doi: 10.7554/eLife.97671

Figure Lengend Snippet: ( A ) Representative images and quantification of IBA1 (cyan)/CD68 (red)-positive microglia in wildtype, Tgfb1 cHet, and Tgfb1 cKO hippocampi (n=3–5; one way ANOVA with Tukey post hoc test; *p<0.05). ( B ) Dotplot of the expression values of TGFB1 signaling components from scRNA-Seq of Tgfb1 WT, cHet, and cKO microglia (n=2 pools of three animals per genotype). ( C ) UMAP plot of Tgfb1 scRNA-Seq colored by genotype. ( D ) FACS plots of gating strategy for microglia isolation for RNA-Seq in cHet and cKO hippocampi. Quantification of the flow cytometry analysis of CD11b and CD45 (n=3–5; mixed effects analysis; *p<0.05, ****p<0.0001). ( E ) Example histogram and quantification of CD48 in control and Tgfb1 cKO FACS-sorted microglia (n=3–5 per group; t -test; ****p<0.0001). ( F ) Top gene ontology terms associated with genes significantly increased in microglia bulk RNA-Seq in Tgfb1 cKO samples (n=3–5 samples per genotype). ( G ) Top gene ontology terms associated with genes significantly decreased in microglia bulk RNA-Seq in Tgfb1 cKO samples. ( H ) Overlap between concordant microglia gene expression changes induced by Tgfb1 knockout and aging. (χ 2 <2.2e-16). ( I ) Heatmap of gene expression of the top 10 genes in each aging module in control and Tgfb1 cKO microglia. ( J ) Average gene expression changes for each aging module represented as log2 fold change of Tgfb1 cKO over control (one-sample t -test with the expected value of 0 [no change]; ****p<0.0001). Data are shown as means ± s.e.m.

Techniques: Expressing, Isolation, RNA Sequencing, Flow Cytometry, Control, Gene Expression, Knock-Out

Journal: eLife

Article Title: Microglia aging in the hippocampus advances through intermediate states that drive activation and cognitive decline

doi: 10.7554/eLife.97671

Figure Lengend Snippet:

Techniques: Software, Magnetic Beads, Recombinant, Sequencing, RNAscope

a , b Representative wound images and wound closure rates of Snhg26 -KO mice ( n = 5) vs. WT mice ( n = 6) ( a) and C57BL/6 mice treated with Snhg26 -ASOs or control ASOs ( n = 5/group) ( b ). Wound healing rate constant κ (day−1) was calculated using a one-phase decay model in GraphPad. c Gene Ontology analysis of differentially expressed genes in the wound-edge epidermis of Snhg26 -KO mice vs. WT mice. d Gene set enrichment analysis of wound-edge epidermal gene expression changes in Snhg26 -KO mice vs. WT mice. Shown are the false discovery rate (FDR) value and normalized enrichment score (NES). e qRT‒PCR analysis of Krt16 and Sprr1b expression in the skin and wound-edge epidermis of Snhg26 -KO and WT mice ( n = 5). f Hematoxylin and eosin staining of Snhg26 -KO and WT mouse wounds. Areas of the wound tongue epithelium (marked with dotted lines) were quantified ( n = 8). Scale bar, 200 μm. g qRT‒PCR analysis of Il6 and Il1b expression in mouse skin and wound-edge epidermis ( n = 5). h Immunofluorescence analysis of CD68 + cells in mouse wounds ( n = 4). The white dashed line demarcates the epidermis and dermis border. Scale bar, 20 μm. Data are shown as mean ± SD from two to three independent experiments ( a , b , e – h ). The data were analyzed by two-way ANOVA ( a , b , e and g ), two- sided Fisher’s exact test ( c ) or two-tailed Student’s t test ( f and h ).

Journal: Nature Communications

Article Title: The lncRNA SNHG26 drives the inflammatory-to-proliferative state transition of keratinocyte progenitor cells during wound healing

doi: 10.1038/s41467-024-52783-8

Figure Lengend Snippet: a , b Representative wound images and wound closure rates of Snhg26 -KO mice ( n = 5) vs. WT mice ( n = 6) ( a) and C57BL/6 mice treated with Snhg26 -ASOs or control ASOs ( n = 5/group) ( b ). Wound healing rate constant κ (day−1) was calculated using a one-phase decay model in GraphPad. c Gene Ontology analysis of differentially expressed genes in the wound-edge epidermis of Snhg26 -KO mice vs. WT mice. d Gene set enrichment analysis of wound-edge epidermal gene expression changes in Snhg26 -KO mice vs. WT mice. Shown are the false discovery rate (FDR) value and normalized enrichment score (NES). e qRT‒PCR analysis of Krt16 and Sprr1b expression in the skin and wound-edge epidermis of Snhg26 -KO and WT mice ( n = 5). f Hematoxylin and eosin staining of Snhg26 -KO and WT mouse wounds. Areas of the wound tongue epithelium (marked with dotted lines) were quantified ( n = 8). Scale bar, 200 μm. g qRT‒PCR analysis of Il6 and Il1b expression in mouse skin and wound-edge epidermis ( n = 5). h Immunofluorescence analysis of CD68 + cells in mouse wounds ( n = 4). The white dashed line demarcates the epidermis and dermis border. Scale bar, 20 μm. Data are shown as mean ± SD from two to three independent experiments ( a , b , e – h ). The data were analyzed by two-way ANOVA ( a , b , e and g ), two- sided Fisher’s exact test ( c ) or two-tailed Student’s t test ( f and h ).

Article Snippet: To check the macrophage infiltration, the rabbit anti-mouse CD68 antibody (1:10,000; MCA1957; AbD Serotec) was applied to the section for overnight at 4 °C.

Techniques: Control, Gene Expression, Expressing, Staining, Immunofluorescence, Two Tailed Test

FPS-ZM1 regulates microglia polarization and reduces SEMA3C-mediated neuroinflammation after SCI 3 mice per group. (A) Representative immunofluorescent images of spinal cord sections 3 days after SCI (3 dpi). The sagittal sections were immunostained for Iba-1 (yellow) and counterstained with DAPI (blue). Low magnification scale bar, 200 μm; high magnification scale bar, 20 μm. (B and C) Quantitation (mean ± SD) of Iba-1+positive cells (B) and Arg-1/CD68 (C) immunostaining showing mean relative fluorescence normalized to the SCI group. For Iba-1+positive cells analysis, F (3, 8) = 417.9, R squared = 0.9937, p value <0.0001 in ANOVA summary. For Arg-1/CD68 (C) immunostaining, F (3, 8) = 416.1, R squared = 0.9936, p value <0.0001 in ANOVA summary. (D) Immunofluorescent images of spinal cord sections at 3 dpi. Sections were immunostained for Arg-1(red)/CD68 (green) and counterstained with DAPI (blue). Scale bars, 200 μm (left panel); 50 μm (right panel). (E) Immunofluorescent images of spinal cord sections at 3 dpi. Sections are immunostained for SEMA3C (green) and counterstained with DAPI (blue). Scale bars, 200 μm (left panel); 50 μm (right panel). (F) Quantitation (mean ± SD) of SEMA3C immunostaining showing mean relative fluorescence intensity normalized to the SCI group. F (3, 8) = 662.8, R squared = 0.9960, p value <0.0001 in ANOVA summary. For all panels with quantitation (B, C, and F), significant differences were evaluated by one-way ANOVA and post hoc Dunnett’s tests; ∗∗∗ p < 0.001 versus the SEMA3C-treated group.

Journal: iScience

Article Title: Semaphorin3C identified as mediator of neuroinflammation and microglia polarization after spinal cord injury

doi: 10.1016/j.isci.2024.109649

Figure Lengend Snippet: FPS-ZM1 regulates microglia polarization and reduces SEMA3C-mediated neuroinflammation after SCI 3 mice per group. (A) Representative immunofluorescent images of spinal cord sections 3 days after SCI (3 dpi). The sagittal sections were immunostained for Iba-1 (yellow) and counterstained with DAPI (blue). Low magnification scale bar, 200 μm; high magnification scale bar, 20 μm. (B and C) Quantitation (mean ± SD) of Iba-1+positive cells (B) and Arg-1/CD68 (C) immunostaining showing mean relative fluorescence normalized to the SCI group. For Iba-1+positive cells analysis, F (3, 8) = 417.9, R squared = 0.9937, p value <0.0001 in ANOVA summary. For Arg-1/CD68 (C) immunostaining, F (3, 8) = 416.1, R squared = 0.9936, p value <0.0001 in ANOVA summary. (D) Immunofluorescent images of spinal cord sections at 3 dpi. Sections were immunostained for Arg-1(red)/CD68 (green) and counterstained with DAPI (blue). Scale bars, 200 μm (left panel); 50 μm (right panel). (E) Immunofluorescent images of spinal cord sections at 3 dpi. Sections are immunostained for SEMA3C (green) and counterstained with DAPI (blue). Scale bars, 200 μm (left panel); 50 μm (right panel). (F) Quantitation (mean ± SD) of SEMA3C immunostaining showing mean relative fluorescence intensity normalized to the SCI group. F (3, 8) = 662.8, R squared = 0.9960, p value <0.0001 in ANOVA summary. For all panels with quantitation (B, C, and F), significant differences were evaluated by one-way ANOVA and post hoc Dunnett’s tests; ∗∗∗ p < 0.001 versus the SEMA3C-treated group.

Article Snippet: Rabbit monoclonal anti-mouse/rat/hamster CD68 , Cell Signaling Technology , Cat# 97778; RRID: AB_2928056.

Techniques: Quantitation Assay, Immunostaining, Fluorescence

FPS-ZM1 improves neuronal survival after SCI (A) Representative immunofluorescent images for TPH2 (green) and GAP43 (red) in sagittal sections and GFAP (green) and GAP43 (red) in transverse sections 14 dpi. t1–t4 refer to different levels of transverse cardinal plane. The four transverse sections are displayed at interval of 1 mm. Scale bars, 1 mm (upper panel); 200 μm (bottom panel). n = 3. (B) Quantitation (mean ± SD) of GAP43+ and GFAP+ positive cells. Significant differences were evaluated by Student’s t test; ∗∗∗ p < 0.001 versus the SEMA3C group. In GAP43+ positive cells analysis, For T1, p value < 0.0001, t = 48.49, df = 4. For T2, p value < 0.0001, t = 23.13, df = 4. For T3, p value < 0.0001, t = 24.98, df = 4. For T4, p value = 0.0004, t = 11.23, df = 4. In GFAP+ positive cells analysis, For T1, p value = 0.1633, t = 1.705, df = 4. For T2, p value = 0.1712, t = 1.665, df = 4. For T3, p value = 0.0036, t = 6.122, df = 4. For T4, p value = 0.2879, t = 1.225, df = 4. (C) Serum serotonin determined by ELISA (mean ± SD). n = 4. Significant differences were evaluated by Student’s t test; ∗ p = 0.0253 versus the SEMA3C group, t = 2.961, df = 6. (D and E) Representative immunofluorescent images for pgp9.5 (green), NeuN (red), and neurofilamment (yellow) in sagittal sections and synaptophysin (green) and NeuN (red) in transverse sections at 14 dpi. Scale bars, 200 μm. Quantitation of NeuN positive cells (Data are represented as mean ± SD). n = 3. Significant differences were evaluated by Student’s t test; ∗∗∗ p = 0.0009 versus the SEMA3C group. t = 8.753, df = 4. (F) Transmission electron microscope images of myelin sheath at 14 days after SCI. Scale bar, 100 μm. Small bar insert, 10μm. n = 5. (G) Quantitative analysis (mean ± SD) for diameter of thickness of myelin sheath. Significant differences were evaluated by one-way ANOVA and post hoc Dunnett’s tests for all panels. F (3, 16) = 5.365, R squared = 0.5015, p value = 0.0095 in ANOVA summary. (H) The gray ratio values (mean ± SD) of RAGE ( p value = 0.0161, t = 4.004, df = 4) and iNOS ( p value = 0.0388, t = 3.029, df = 4) to βactin are shown. WT versus KO mice. n = 3. Significant differences were evaluated by Student’s t test; ∗ p < 0.05 versus the WT group. (I) Representative immunofluorescent images for CD68 (red) in WT and KO mice. Scale bar, 200 μm.

Journal: iScience

Article Title: Semaphorin3C identified as mediator of neuroinflammation and microglia polarization after spinal cord injury

doi: 10.1016/j.isci.2024.109649

Figure Lengend Snippet: FPS-ZM1 improves neuronal survival after SCI (A) Representative immunofluorescent images for TPH2 (green) and GAP43 (red) in sagittal sections and GFAP (green) and GAP43 (red) in transverse sections 14 dpi. t1–t4 refer to different levels of transverse cardinal plane. The four transverse sections are displayed at interval of 1 mm. Scale bars, 1 mm (upper panel); 200 μm (bottom panel). n = 3. (B) Quantitation (mean ± SD) of GAP43+ and GFAP+ positive cells. Significant differences were evaluated by Student’s t test; ∗∗∗ p < 0.001 versus the SEMA3C group. In GAP43+ positive cells analysis, For T1, p value < 0.0001, t = 48.49, df = 4. For T2, p value < 0.0001, t = 23.13, df = 4. For T3, p value < 0.0001, t = 24.98, df = 4. For T4, p value = 0.0004, t = 11.23, df = 4. In GFAP+ positive cells analysis, For T1, p value = 0.1633, t = 1.705, df = 4. For T2, p value = 0.1712, t = 1.665, df = 4. For T3, p value = 0.0036, t = 6.122, df = 4. For T4, p value = 0.2879, t = 1.225, df = 4. (C) Serum serotonin determined by ELISA (mean ± SD). n = 4. Significant differences were evaluated by Student’s t test; ∗ p = 0.0253 versus the SEMA3C group, t = 2.961, df = 6. (D and E) Representative immunofluorescent images for pgp9.5 (green), NeuN (red), and neurofilamment (yellow) in sagittal sections and synaptophysin (green) and NeuN (red) in transverse sections at 14 dpi. Scale bars, 200 μm. Quantitation of NeuN positive cells (Data are represented as mean ± SD). n = 3. Significant differences were evaluated by Student’s t test; ∗∗∗ p = 0.0009 versus the SEMA3C group. t = 8.753, df = 4. (F) Transmission electron microscope images of myelin sheath at 14 days after SCI. Scale bar, 100 μm. Small bar insert, 10μm. n = 5. (G) Quantitative analysis (mean ± SD) for diameter of thickness of myelin sheath. Significant differences were evaluated by one-way ANOVA and post hoc Dunnett’s tests for all panels. F (3, 16) = 5.365, R squared = 0.5015, p value = 0.0095 in ANOVA summary. (H) The gray ratio values (mean ± SD) of RAGE ( p value = 0.0161, t = 4.004, df = 4) and iNOS ( p value = 0.0388, t = 3.029, df = 4) to βactin are shown. WT versus KO mice. n = 3. Significant differences were evaluated by Student’s t test; ∗ p < 0.05 versus the WT group. (I) Representative immunofluorescent images for CD68 (red) in WT and KO mice. Scale bar, 200 μm.

Article Snippet: Rabbit monoclonal anti-mouse/rat/hamster CD68 , Cell Signaling Technology , Cat# 97778; RRID: AB_2928056.

Techniques: Quantitation Assay, Enzyme-linked Immunosorbent Assay, Transmission Assay, Microscopy

Journal: iScience

Article Title: Semaphorin3C identified as mediator of neuroinflammation and microglia polarization after spinal cord injury

doi: 10.1016/j.isci.2024.109649

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-mouse/rat/hamster CD68 , Cell Signaling Technology , Cat# 97778; RRID: AB_2928056.

Techniques: Ubiquitin Proteomics, Recombinant, Purification, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, In Situ Hybridization, Negative Control, Software

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