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FUJIFILM rabbit anti iba1
Lipid droplet accumulating microglia are abundant in the hippocampus but rare in other brain regions of aged mice. a – d , Representative confocal images of the cortex ( a ), thalamus ( b ), corpus callosum ( c ) and hippocampal dentate gyrus ( d ) from 20-month old male mice stained for BODIPY + (lipid droplets) and <t>Iba1</t> + (microglia). Scale bar: 20 μm. Arrows point towards BODIPY + lipid droplets. e, Quantification of BODIPY+/Iba1+ cells. n = 4 mice per group. One-way ANOVA followed by Tukey’s post hoc test. Error bars represent mean ± SD. ***P
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1) Product Images from "Lipid droplet accumulating microglia represent a dysfunctional and pro-inflammatory state in the aging brain"

Article Title: Lipid droplet accumulating microglia represent a dysfunctional and pro-inflammatory state in the aging brain

Journal: Nature neuroscience

doi: 10.1038/s41593-019-0566-1

Lipid droplet accumulating microglia are abundant in the hippocampus but rare in other brain regions of aged mice. a – d , Representative confocal images of the cortex ( a ), thalamus ( b ), corpus callosum ( c ) and hippocampal dentate gyrus ( d ) from 20-month old male mice stained for BODIPY + (lipid droplets) and Iba1 + (microglia). Scale bar: 20 μm. Arrows point towards BODIPY + lipid droplets. e, Quantification of BODIPY+/Iba1+ cells. n = 4 mice per group. One-way ANOVA followed by Tukey’s post hoc test. Error bars represent mean ± SD. ***P
Figure Legend Snippet: Lipid droplet accumulating microglia are abundant in the hippocampus but rare in other brain regions of aged mice. a – d , Representative confocal images of the cortex ( a ), thalamus ( b ), corpus callosum ( c ) and hippocampal dentate gyrus ( d ) from 20-month old male mice stained for BODIPY + (lipid droplets) and Iba1 + (microglia). Scale bar: 20 μm. Arrows point towards BODIPY + lipid droplets. e, Quantification of BODIPY+/Iba1+ cells. n = 4 mice per group. One-way ANOVA followed by Tukey’s post hoc test. Error bars represent mean ± SD. ***P

Techniques Used: Mouse Assay, Staining

Lipid droplet containing microglia in the cortex, corpus callosum, and thalamus of GRN −/− mice. a - c , Representative confocal images of BODIPY + (lipid droplets) and Iba1 + (microglia) in the cortex ( a ), corpus callosum ( b ), ( c ) and thalamus from 9-month-old male GRN −/− mice. BODIPY + /Iba1 + cells were frequently found in the thalamus and were detected to a lesser extent in cortex and corpus callosum. Data were replicated in at least three independent experiments.
Figure Legend Snippet: Lipid droplet containing microglia in the cortex, corpus callosum, and thalamus of GRN −/− mice. a - c , Representative confocal images of BODIPY + (lipid droplets) and Iba1 + (microglia) in the cortex ( a ), corpus callosum ( b ), ( c ) and thalamus from 9-month-old male GRN −/− mice. BODIPY + /Iba1 + cells were frequently found in the thalamus and were detected to a lesser extent in cortex and corpus callosum. Data were replicated in at least three independent experiments.

Techniques Used: Mouse Assay

LPS treatment induces lipid droplet formation in microglia and in BV2 cells. a , b , 3-month-old male mice were given intraperitoneal (i.p.) injections of LPS (1 mg/kg BW) for four days. Representative confocal images of BODIPY + and Tmem119 + in the hippocampus ( a ) and of BODIPY and Iba1 staining in the cortex, corpus callosum, and thalamus ( b ). c - e , Lipidome profiling of lipid droplets from LPS-treated BV2 cells, primary microglia, and liver tissue. c , Pie charts showing that the lipid composition of lipid droplets from young and aged microglia is highly similar, but differs between young and aged liver tissue. d , e , Distribution of MAG chain lengths ( d ) and TAG saturation levels ( e ) of lipid droplets isolated from LPS-treated BV2 cells and from microglia and liver tissue from aged mice. young = 5-month-old male mice, old= 20-month-old male mice; n = 4 mice per group. Data in a-b were replicated in at least two independent experiments. Error bars represent mean ± s.e.m. Scale bars, 20 μm.
Figure Legend Snippet: LPS treatment induces lipid droplet formation in microglia and in BV2 cells. a , b , 3-month-old male mice were given intraperitoneal (i.p.) injections of LPS (1 mg/kg BW) for four days. Representative confocal images of BODIPY + and Tmem119 + in the hippocampus ( a ) and of BODIPY and Iba1 staining in the cortex, corpus callosum, and thalamus ( b ). c - e , Lipidome profiling of lipid droplets from LPS-treated BV2 cells, primary microglia, and liver tissue. c , Pie charts showing that the lipid composition of lipid droplets from young and aged microglia is highly similar, but differs between young and aged liver tissue. d , e , Distribution of MAG chain lengths ( d ) and TAG saturation levels ( e ) of lipid droplets isolated from LPS-treated BV2 cells and from microglia and liver tissue from aged mice. young = 5-month-old male mice, old= 20-month-old male mice; n = 4 mice per group. Data in a-b were replicated in at least two independent experiments. Error bars represent mean ± s.e.m. Scale bars, 20 μm.

Techniques Used: Mouse Assay, Staining, Isolation

Microglia in the aged brain accumulate lipid droplets. a , Electron microscopy of microglia from young and aged mice. b , Hippocampus from aged mice stained for BODIPY + (lipid droplets) and TMEM119 + (microglia). Right panel shows 3D reconstruction of BODIPY + /TMEM119 + microglia. Arrows indicate lipid droplets. c-e , Quantification of BODIPY + lipid droplet numbers ( c ), percent BODIPY + /TMEM119 + cells ( d ), and average BODIPY + lipid droplet size ( e ) in the hippocampus (dentate gyrus). n = 6 mice per group. f , Representative image of Plin3 + (lipid droplets) TMEM119 + microglia in aged mice. g , Confocal images of Plin2 + (lipid droplets) and Iba1 + (microglia) in the human hippocampus of a 22-year-old and 67-year-old individual. Arrows indicate Plin2 + Iba1 + cells. h , i , Representative images ( h ) and quantification ( i; P=0.01) of CARS + signal (2845 cm −1 ) in TMEM119 + microglia in the hippocampus of young and aged mice. n = 5 mice per group. j , k Experimental schematic for lipidomics analysis of lipid droplets isolated from whole hippocampus and from FACS-sorted microglia from 20-month old mice ( j) , and pie charts showing the composition of lipid droplets ( k ); n = 4 mice per group. Statistical tests: two-sided Student’s t-test. Error bars represent mean ± SD. **P
Figure Legend Snippet: Microglia in the aged brain accumulate lipid droplets. a , Electron microscopy of microglia from young and aged mice. b , Hippocampus from aged mice stained for BODIPY + (lipid droplets) and TMEM119 + (microglia). Right panel shows 3D reconstruction of BODIPY + /TMEM119 + microglia. Arrows indicate lipid droplets. c-e , Quantification of BODIPY + lipid droplet numbers ( c ), percent BODIPY + /TMEM119 + cells ( d ), and average BODIPY + lipid droplet size ( e ) in the hippocampus (dentate gyrus). n = 6 mice per group. f , Representative image of Plin3 + (lipid droplets) TMEM119 + microglia in aged mice. g , Confocal images of Plin2 + (lipid droplets) and Iba1 + (microglia) in the human hippocampus of a 22-year-old and 67-year-old individual. Arrows indicate Plin2 + Iba1 + cells. h , i , Representative images ( h ) and quantification ( i; P=0.01) of CARS + signal (2845 cm −1 ) in TMEM119 + microglia in the hippocampus of young and aged mice. n = 5 mice per group. j , k Experimental schematic for lipidomics analysis of lipid droplets isolated from whole hippocampus and from FACS-sorted microglia from 20-month old mice ( j) , and pie charts showing the composition of lipid droplets ( k ); n = 4 mice per group. Statistical tests: two-sided Student’s t-test. Error bars represent mean ± SD. **P

Techniques Used: Electron Microscopy, Mouse Assay, Staining, Isolation, FACS

LPS treatment induces lipid droplet formation in microglia. a-d , BV2 cells were treated with PBS or LPS (5 μg/ml) for 18 hours and co-treated with Triacsin C (1μM) or saline. Representative micrographs of BODIPY + staining in BV2 cells ( a ) and quantification of BODIPY + cells ( b ). c , d , Representative flow cytometry histogram ( c ) and quantification ( d ) of BODIPY fluorescence in BV2 cells. e - h , Lipidome profiling of lipid droplets from LPS-treated BV2 cells, and from primary microglia and liver tissue from aged (20-month-old) mice. Overall composition of lipid droplets ( e ), percentage of neutral lipids ( f ), and distribution of TAG species ( g , h ). i , j , BODIPY + and Iba1 + in the hippocampus of 3-month old male mice given intraperitoneal (i.p.) injections of PBS or LPS (1 mg/kg BW) for four days. Representative confocal images ( i ) and quantification ( j ; P=0.008) of BODIPY + /Iba1 + microglia. n = 6 mice per group. k - m , RNA-Sequencing of BODIPY lo (=LD-lo) and BODIPY hi (=LD-hi) CD11b + CD45 lo microglia from the hippocampus of 3-month old LPS-treated mice. n = 3 biologically independent samples per group. Each sample is a pool of microglia from the hippocampi of two mice. k , Volcano plot showing differentially expressed genes in LD-hi versus LD-lo microglia. Dashed line represents q-value
Figure Legend Snippet: LPS treatment induces lipid droplet formation in microglia. a-d , BV2 cells were treated with PBS or LPS (5 μg/ml) for 18 hours and co-treated with Triacsin C (1μM) or saline. Representative micrographs of BODIPY + staining in BV2 cells ( a ) and quantification of BODIPY + cells ( b ). c , d , Representative flow cytometry histogram ( c ) and quantification ( d ) of BODIPY fluorescence in BV2 cells. e - h , Lipidome profiling of lipid droplets from LPS-treated BV2 cells, and from primary microglia and liver tissue from aged (20-month-old) mice. Overall composition of lipid droplets ( e ), percentage of neutral lipids ( f ), and distribution of TAG species ( g , h ). i , j , BODIPY + and Iba1 + in the hippocampus of 3-month old male mice given intraperitoneal (i.p.) injections of PBS or LPS (1 mg/kg BW) for four days. Representative confocal images ( i ) and quantification ( j ; P=0.008) of BODIPY + /Iba1 + microglia. n = 6 mice per group. k - m , RNA-Sequencing of BODIPY lo (=LD-lo) and BODIPY hi (=LD-hi) CD11b + CD45 lo microglia from the hippocampus of 3-month old LPS-treated mice. n = 3 biologically independent samples per group. Each sample is a pool of microglia from the hippocampi of two mice. k , Volcano plot showing differentially expressed genes in LD-hi versus LD-lo microglia. Dashed line represents q-value

Techniques Used: Staining, Flow Cytometry, Fluorescence, Mouse Assay, RNA Sequencing Assay

LDAM and lipid droplets in BV2 cells are associated with impaired phagocytosis. a , Pathway map of genes related to phagosome maturation that are differentially expressed in LDAM (see Fig. 2 ). b , Confocal images showing BODIPY + (lipid droplets), CD68 + (endosomes/ lysosomes), and Iba1 + in the hippocampus from 20-month old mice. c , Percentage of BODIPY - and BODIPY + Iba1 + microglia with high levels of CD68 (CD68 hi ). n = 5 mice per group; P=0.002. d , 3D reconstruction of Iba1 + microglia showing BODIPY + lipid droplets closely surrounded by CD68 + vesicles. e , Electron microscopy image showing lysosomal accumulation in LDAM from a 20-month old mouse. f , g , Confocal images ( f ) and quantification ( g ) of BODIPY + and Zymosan + in BV2 cells treated with LPS (5 ug/ml) for 18 hours. h , i , Phagocytosis of pHrodo red Zymosan in BV2 cells treated with PBS or LPS and co-treated with Triacsin C (1μM) or saline. Representative images of ( h ) and time lapse imaging and quantification ( i ) of Zymosan uptake in BV2 cells. j , k , 250 μm organotypic brain slices from 12-month old mice were incubated for 4 hours with pHrodo red Zymosan particles. Representative confocal images of the hippocampus ( j ) and pie chart showing the percentages of Zymosan-containing BODIPY - and BODIPY + Iba1 + cells ( k ). P-value for Zymosan + BODIPY - vs Zymosan + BODIPY + Iba1 + cells = 0.0012. n = 3 mice per group. l-n , Myelin debris labelled with Alexa Fluor 555 was stereotactically injected into the hippocampus of 20-month old male mice, and phagocytosis was analyzed 48 hours after injection. m , Representative images of AF555-labelled myelin (left panel) and of Iba1 + cells with and without lipid droplets (BODIPY + ) and A555 + myelin. n , Quantification of myelin uptake in BODIPY + /Iba1 + and BODIPY - Iba1 + cells. n = 6 mice per group; P=0.038. Experiments on BV2 cells were performed three times in technical triplicates. Statistical tests: two-sided Student’s t-test (c,g,n), two-way ANOVA followed by Tukey’s post hoc test (i,k). Error bars represent mean ± SD (c,g,n) and mean ± SEM (i). *P
Figure Legend Snippet: LDAM and lipid droplets in BV2 cells are associated with impaired phagocytosis. a , Pathway map of genes related to phagosome maturation that are differentially expressed in LDAM (see Fig. 2 ). b , Confocal images showing BODIPY + (lipid droplets), CD68 + (endosomes/ lysosomes), and Iba1 + in the hippocampus from 20-month old mice. c , Percentage of BODIPY - and BODIPY + Iba1 + microglia with high levels of CD68 (CD68 hi ). n = 5 mice per group; P=0.002. d , 3D reconstruction of Iba1 + microglia showing BODIPY + lipid droplets closely surrounded by CD68 + vesicles. e , Electron microscopy image showing lysosomal accumulation in LDAM from a 20-month old mouse. f , g , Confocal images ( f ) and quantification ( g ) of BODIPY + and Zymosan + in BV2 cells treated with LPS (5 ug/ml) for 18 hours. h , i , Phagocytosis of pHrodo red Zymosan in BV2 cells treated with PBS or LPS and co-treated with Triacsin C (1μM) or saline. Representative images of ( h ) and time lapse imaging and quantification ( i ) of Zymosan uptake in BV2 cells. j , k , 250 μm organotypic brain slices from 12-month old mice were incubated for 4 hours with pHrodo red Zymosan particles. Representative confocal images of the hippocampus ( j ) and pie chart showing the percentages of Zymosan-containing BODIPY - and BODIPY + Iba1 + cells ( k ). P-value for Zymosan + BODIPY - vs Zymosan + BODIPY + Iba1 + cells = 0.0012. n = 3 mice per group. l-n , Myelin debris labelled with Alexa Fluor 555 was stereotactically injected into the hippocampus of 20-month old male mice, and phagocytosis was analyzed 48 hours after injection. m , Representative images of AF555-labelled myelin (left panel) and of Iba1 + cells with and without lipid droplets (BODIPY + ) and A555 + myelin. n , Quantification of myelin uptake in BODIPY + /Iba1 + and BODIPY - Iba1 + cells. n = 6 mice per group; P=0.038. Experiments on BV2 cells were performed three times in technical triplicates. Statistical tests: two-sided Student’s t-test (c,g,n), two-way ANOVA followed by Tukey’s post hoc test (i,k). Error bars represent mean ± SD (c,g,n) and mean ± SEM (i). *P

Techniques Used: Mouse Assay, Electron Microscopy, Imaging, Incubation, Injection

GRN −/− mice possess high numbers of lipid droplet-rich microglia which functionally and partially transcriptionally resemble LDAM. a - c , BODIPY + and Iba1 + expression in the hippocampus of 9-month old male WT mice and age- and sex-matched GRN −/− mice. Representative confocal images ( a ), and quantification of BODIPY + /Iba1 + microglia ( b , P=0.0064) and of lipid droplet numbers per Iba1 + microglia ( c ). n = 5 mice per group. d , e , 250 μm organotypic brain slices from 9-month old GRN −/− mice were incubated for 4 hours with pHrodo red Zymosan particles. Representative confocal images of the hippocampus ( d ) and quantification of Zymosan uptake in BODIPY - and BODIPY + Iba1 + cells ( e ). n = 3 mice per group. f , Quantification of CellROX fluorescence in primary LD-lo and LD-hi microglia from 9-month old GRN −/− mice and age-matched wild type controls. g , Acutely isolated LD-lo and LD-hi primary microglia from 10-month old GRN −/− mice were treated with LPS (100 ng/ml) or PBS for 8 hours. Heatmap shows changes in cytokine secretion under baseline conditions (PBS) and after LPS treatment. h - k , RNA-Sequencing of BODIPY lo (=LD-lo) and BODIPY hi (=LD-hi) CD11b + CD45 lo microglia from the hippocampus of 10-month old GRN −/− mice. n = 3 biologically independent samples per group. Each sample is a pool of microglia from the hippocampi of two mice. h , Volcano plot showing differentially expressed genes in LD-hi versus LD-lo microglia. Dashed line represents q-value
Figure Legend Snippet: GRN −/− mice possess high numbers of lipid droplet-rich microglia which functionally and partially transcriptionally resemble LDAM. a - c , BODIPY + and Iba1 + expression in the hippocampus of 9-month old male WT mice and age- and sex-matched GRN −/− mice. Representative confocal images ( a ), and quantification of BODIPY + /Iba1 + microglia ( b , P=0.0064) and of lipid droplet numbers per Iba1 + microglia ( c ). n = 5 mice per group. d , e , 250 μm organotypic brain slices from 9-month old GRN −/− mice were incubated for 4 hours with pHrodo red Zymosan particles. Representative confocal images of the hippocampus ( d ) and quantification of Zymosan uptake in BODIPY - and BODIPY + Iba1 + cells ( e ). n = 3 mice per group. f , Quantification of CellROX fluorescence in primary LD-lo and LD-hi microglia from 9-month old GRN −/− mice and age-matched wild type controls. g , Acutely isolated LD-lo and LD-hi primary microglia from 10-month old GRN −/− mice were treated with LPS (100 ng/ml) or PBS for 8 hours. Heatmap shows changes in cytokine secretion under baseline conditions (PBS) and after LPS treatment. h - k , RNA-Sequencing of BODIPY lo (=LD-lo) and BODIPY hi (=LD-hi) CD11b + CD45 lo microglia from the hippocampus of 10-month old GRN −/− mice. n = 3 biologically independent samples per group. Each sample is a pool of microglia from the hippocampi of two mice. h , Volcano plot showing differentially expressed genes in LD-hi versus LD-lo microglia. Dashed line represents q-value

Techniques Used: Mouse Assay, Expressing, Incubation, Fluorescence, Isolation, RNA Sequencing Assay

2) Product Images from "Suppressing pro-inflammatory prostaglandin signaling attenuates excitotoxicity-associated neuronal inflammation and injury"

Article Title: Suppressing pro-inflammatory prostaglandin signaling attenuates excitotoxicity-associated neuronal inflammation and injury

Journal: Neuropharmacology

doi: 10.1016/j.neuropharm.2019.02.011

Post-SE treatment with EP2 antagonist mitigates seizure-induced glial activation. Three days after kainate-induced SE, the expression of biomarkers GFAP for astrocyte activation and Iba1 for microglia in the mouse hippocampus were measured by qPCR for their mRNA levels (A) and western blot for protein levels (B) (* P
Figure Legend Snippet: Post-SE treatment with EP2 antagonist mitigates seizure-induced glial activation. Three days after kainate-induced SE, the expression of biomarkers GFAP for astrocyte activation and Iba1 for microglia in the mouse hippocampus were measured by qPCR for their mRNA levels (A) and western blot for protein levels (B) (* P

Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

3) Product Images from "Pre-treatment with meloxicam prevents the spinal inflammation and oxidative stress in DRG neurons that accompany painful cervical radiculopathy"

Article Title: Pre-treatment with meloxicam prevents the spinal inflammation and oxidative stress in DRG neurons that accompany painful cervical radiculopathy

Journal: Neuroscience

doi: 10.1016/j.neuroscience.2018.07.054

Representative images of the C7 spinal dorsal horn at day 7 showing that the oxidative stress marker 8-OHG does not colocalize with Iba1 or GFAP but does colocalize with the neuronal marker, NeuN. Enlarged images show colocalization of 8-OHG and NeuN in the superficial dorsal horn for each study group. Scale bar in the full size images is 100¼m and is 50μm in the insets of the superficial dorsal horn.
Figure Legend Snippet: Representative images of the C7 spinal dorsal horn at day 7 showing that the oxidative stress marker 8-OHG does not colocalize with Iba1 or GFAP but does colocalize with the neuronal marker, NeuN. Enlarged images show colocalization of 8-OHG and NeuN in the superficial dorsal horn for each study group. Scale bar in the full size images is 100¼m and is 50μm in the insets of the superficial dorsal horn.

Techniques Used: Marker

Pre-treatment with meloxicam before NRC prevents upregulation of spinal sPLA2. Representative images in the spinal dorsal horn show less expression of sPLA2 (green) with meloxicam treatment than NRC, and similar to sham and normal levels. Similarly, there is less colocalization (yellow) evident between sPLA2 (green) and each of MAP2, Iba1 and GFAP (red) in the meloxicam treated (MxNRC), sham and normal groups compared to the NRC group. Spinal sPLA2 expression in the NRC group is significantly higher (*p
Figure Legend Snippet: Pre-treatment with meloxicam before NRC prevents upregulation of spinal sPLA2. Representative images in the spinal dorsal horn show less expression of sPLA2 (green) with meloxicam treatment than NRC, and similar to sham and normal levels. Similarly, there is less colocalization (yellow) evident between sPLA2 (green) and each of MAP2, Iba1 and GFAP (red) in the meloxicam treated (MxNRC), sham and normal groups compared to the NRC group. Spinal sPLA2 expression in the NRC group is significantly higher (*p

Techniques Used: Expressing

Meloxicam injection prevents the microglial and astrocytic activation that is typically observed after NRC in the superficial dorsal horn at day 7. Representative images show Iba1 (red) and GFAP (green) labeling in the ipsilateral dorsal horn of the C7 spinal cord. In the MxNRC group, Iba1 labeling remains at sham and normal levels and is significantly lower than levels in the NRC group (*p
Figure Legend Snippet: Meloxicam injection prevents the microglial and astrocytic activation that is typically observed after NRC in the superficial dorsal horn at day 7. Representative images show Iba1 (red) and GFAP (green) labeling in the ipsilateral dorsal horn of the C7 spinal cord. In the MxNRC group, Iba1 labeling remains at sham and normal levels and is significantly lower than levels in the NRC group (*p

Techniques Used: Injection, Activation Assay, Labeling

4) Product Images from "Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain"

Article Title: Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain

Journal: PLoS ONE

doi: 10.1371/journal.pone.0151589

Microglial response to graphene-free or—inclusive PCL scaffold implantation at Week 1 and Week 3. (A) Image shows an overview of microglial infiltration into the outer shell of a gP6 scaffold at Week 3, and gP6 at high magnification at (B) Week 1 and (C) Week 3. (D) Graphene-free P6 scaffold at Week 3 (Green: Iba1 + microglia cells, blue: DAPI stained nucleus). (E) Microglial profile across the tissue/scaffold interfaces (*** p
Figure Legend Snippet: Microglial response to graphene-free or—inclusive PCL scaffold implantation at Week 1 and Week 3. (A) Image shows an overview of microglial infiltration into the outer shell of a gP6 scaffold at Week 3, and gP6 at high magnification at (B) Week 1 and (C) Week 3. (D) Graphene-free P6 scaffold at Week 3 (Green: Iba1 + microglia cells, blue: DAPI stained nucleus). (E) Microglial profile across the tissue/scaffold interfaces (*** p

Techniques Used: Staining

5) Product Images from "Gelatinase Biosensor Reports Cellular Remodeling During Epileptogenesis"

Article Title: Gelatinase Biosensor Reports Cellular Remodeling During Epileptogenesis

Journal: Frontiers in Synaptic Neuroscience

doi: 10.3389/fnsyn.2020.00015

ACPPs reveal cell-type-specific kinetics of gelatinase activity during epileptogenesis. The gelatinase spatiotemporal activation profile was observed with gelatinase-ACPP uptake in CA1 and molecular and granular layers of KA-injected hippocampi at 24 h, 7 days after KA injection or during the chronic phase. Slices were stained with neurons marker NeuN (A–C,J–L ), microglial marker Iba1 (D–F,M–O ) or astrocytic marker GFAP (G–I,P–R ). Merge of the cell type marker (green) and TAMRA ACPP (orange) are shown. n = 3 independent experiments, two or three animals injected per condition for each experiment, three slices per mouse were used. Scale bar 20 μm.
Figure Legend Snippet: ACPPs reveal cell-type-specific kinetics of gelatinase activity during epileptogenesis. The gelatinase spatiotemporal activation profile was observed with gelatinase-ACPP uptake in CA1 and molecular and granular layers of KA-injected hippocampi at 24 h, 7 days after KA injection or during the chronic phase. Slices were stained with neurons marker NeuN (A–C,J–L ), microglial marker Iba1 (D–F,M–O ) or astrocytic marker GFAP (G–I,P–R ). Merge of the cell type marker (green) and TAMRA ACPP (orange) are shown. n = 3 independent experiments, two or three animals injected per condition for each experiment, three slices per mouse were used. Scale bar 20 μm.

Techniques Used: Activity Assay, Activation Assay, Injection, Staining, Marker

6) Product Images from "Lactate receptor HCAR1 regulates neurogenesis and microglia activation after neonatal hypoxia-ischemia"

Article Title: Lactate receptor HCAR1 regulates neurogenesis and microglia activation after neonatal hypoxia-ischemia

Journal: bioRxiv

doi: 10.1101/2020.12.02.408070

HCAR1 KO mice have deficient activation and proliferation of microglia after HI a-d Confocal images from the peri-infarct zone (b, d, indicated as square in cartoon) and corresponding contralateral area (a, c) of coronal mouse brain sections from WT (a-b) and KO (c-d) labelled for BrdU (proliferating cells, magenta), Iba1 (microglia, green) and DAPI (blue nuclei). Scale bars are 100 μm. e Density of microglia (IBA1+ cells) in the peri-infarct zone (pink bars) and contralateral striatum (white bars) of WT and KO mice. f Density of proliferating microglia (i.e. cells that were both IBA1+ and BrdU+). g-h Assessment of microglia activation by morphology. When activated, microglia somata increase in size and get shorter and fewer branches. g average size of microglia somata. h Average maximum branch length. *p
Figure Legend Snippet: HCAR1 KO mice have deficient activation and proliferation of microglia after HI a-d Confocal images from the peri-infarct zone (b, d, indicated as square in cartoon) and corresponding contralateral area (a, c) of coronal mouse brain sections from WT (a-b) and KO (c-d) labelled for BrdU (proliferating cells, magenta), Iba1 (microglia, green) and DAPI (blue nuclei). Scale bars are 100 μm. e Density of microglia (IBA1+ cells) in the peri-infarct zone (pink bars) and contralateral striatum (white bars) of WT and KO mice. f Density of proliferating microglia (i.e. cells that were both IBA1+ and BrdU+). g-h Assessment of microglia activation by morphology. When activated, microglia somata increase in size and get shorter and fewer branches. g average size of microglia somata. h Average maximum branch length. *p

Techniques Used: Mouse Assay, Activation Assay

7) Product Images from "Loss of OMA1 delays neurodegeneration by preventing stress-induced OPA1 processing in mitochondria"

Article Title: Loss of OMA1 delays neurodegeneration by preventing stress-induced OPA1 processing in mitochondria

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201507022

Oma1 deletion in Phb2 NKO mice prevents neuroinflammation. (A) Reactive astrogliosis and activation of microglia in Phb2 NKO mice is blocked by deletion of Oma1 . Immunohistochemical staining of coronal sections across the hippocampal region from 14-wk-old mice using GFAP- and IBA1-specific antibodies. Bars, 200 µm. (B) Immunoblot analysis of hippocampal lysates from 14-wk-old mice. GAPDH was used as a loading control. (C) mRNA levels of proinflammatory cytokines in Phb2 NKO mice are restored upon deletion of Oma1 . Total RNA was isolated from the hippocampus of 14-wk-old mice and subjected to quantitative real-time PCR analysis ( n = 3). Transcript levels were normalized to Gapdh mRNA levels. **, P
Figure Legend Snippet: Oma1 deletion in Phb2 NKO mice prevents neuroinflammation. (A) Reactive astrogliosis and activation of microglia in Phb2 NKO mice is blocked by deletion of Oma1 . Immunohistochemical staining of coronal sections across the hippocampal region from 14-wk-old mice using GFAP- and IBA1-specific antibodies. Bars, 200 µm. (B) Immunoblot analysis of hippocampal lysates from 14-wk-old mice. GAPDH was used as a loading control. (C) mRNA levels of proinflammatory cytokines in Phb2 NKO mice are restored upon deletion of Oma1 . Total RNA was isolated from the hippocampus of 14-wk-old mice and subjected to quantitative real-time PCR analysis ( n = 3). Transcript levels were normalized to Gapdh mRNA levels. **, P

Techniques Used: Mouse Assay, Activation Assay, Immunohistochemistry, Staining, Isolation, Real-time Polymerase Chain Reaction

8) Product Images from "Loss of astrocyte connexins 43 and 30 does not significantly alter susceptibility or severity of acute experimental autoimmune encephalomyelitis in mice"

Article Title: Loss of astrocyte connexins 43 and 30 does not significantly alter susceptibility or severity of acute experimental autoimmune encephalomyelitis in mice

Journal: Journal of neuroimmunology

doi: 10.1016/j.jneuroim.2012.01.007

Leukocyte and microglial immunostaining in spinal cord sections from WT and Cx43 sKO EAE mice Sections were stained for CD45 (panels A C) or Iba1 (panel E-G) and the extent of immunoreactivity was quantified using ImageJ (D, H). Panels A-B , low power images from WT (A) and Cx43 sKO (B) mice exhibit CD45+ leukocytes in inflammatory lesions. The submeningeal location in white matter is characteristic of lesion distribution in EAE. C, A higher power view of the lateral column from the Cx43 sKO mouse shows perivascular cuffing (arrows) and parenchymal infiltration in an EAE lesion. D, quantification of immunostaining showed no difference between WT (n=9) and Cx43 sKO (n=7) EAE mice. E-F , serial sections stained for the microglial calcium-binding protein Iba1. Low power view (4x) shows strong immunostaining in submeningeal lesions and gray matter. G, A higher power (20x) view shows that immunoreactivity is associated with small process bearing cells consistent with a microglial phenotype. Serial section from panel C. H, quantification of immunostaining showed no difference in EAE between WT and Cx43 sKO mice.
Figure Legend Snippet: Leukocyte and microglial immunostaining in spinal cord sections from WT and Cx43 sKO EAE mice Sections were stained for CD45 (panels A C) or Iba1 (panel E-G) and the extent of immunoreactivity was quantified using ImageJ (D, H). Panels A-B , low power images from WT (A) and Cx43 sKO (B) mice exhibit CD45+ leukocytes in inflammatory lesions. The submeningeal location in white matter is characteristic of lesion distribution in EAE. C, A higher power view of the lateral column from the Cx43 sKO mouse shows perivascular cuffing (arrows) and parenchymal infiltration in an EAE lesion. D, quantification of immunostaining showed no difference between WT (n=9) and Cx43 sKO (n=7) EAE mice. E-F , serial sections stained for the microglial calcium-binding protein Iba1. Low power view (4x) shows strong immunostaining in submeningeal lesions and gray matter. G, A higher power (20x) view shows that immunoreactivity is associated with small process bearing cells consistent with a microglial phenotype. Serial section from panel C. H, quantification of immunostaining showed no difference in EAE between WT and Cx43 sKO mice.

Techniques Used: Immunostaining, Mouse Assay, Staining, Binding Assay

9) Product Images from "RNS60 exerts therapeutic effects in the SOD1 ALS mouse model through protective glia and peripheral nerve rescue"

Article Title: RNS60 exerts therapeutic effects in the SOD1 ALS mouse model through protective glia and peripheral nerve rescue

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-018-1101-0

RNS60 activates astrocytes and phagocytic microglia and increases anti-inflammatory molecules in C57BL/6-SOD1 G93A mice. a – c Representative images of ventral LSC hemisections stained with GFAP (green) at 20 weeks. Scale bar: 50 μm. d Quantification of GFAP immunofluorescence showed elevated astrocytosis in the LSC ventral horn of C57BL/6-SOD1 G93A mice at the symptomatic stage of the pathology as compared to NTG expressed as 100%. Treatment with RNS60 significantly increased reactive astrocytosis. Bars are mean ± SEM as a percentage versus NTG controls ( n = 3 animals for each group). e – g IBA1-stained (light blue) LSC hemisection from NTG, and SOD1 G93A mice treated with NS or RNS60. Scale bar: 50 μm. h Relative quantification showed higher level of IBA1 immunofluorescence in transgenic mice compared to NTG, without changes after the treatment with RNS60. Data are expressed as mean ± SEM as percentage versus NTG ( n = 3 animals for each group). i – k CD68 positive macrophagic microglia (red) was labeled in ventral horn of LSC. Scale bar: 50 μm. l Immunoreactivity quantification showed an up-regulation of CD68 in the LSC of C57BL/6-SOD1G93A mice compared to NTG. This effect was exacerbated by the treatment with RNS60. The bar graph represents mean ± SEM as a percentage versus NTG controls ( n = 3 mice per group). m – p Real-time PCR for pro-inflammatory (CCL2 and IL-1β) and anti-inflammatory (IL-4 and YM1) markers in the ventral portion of LSC of NTG mice expressed as 100% and transgenic mice treated with NS or RNS60. For CCL2 ( p = 0.7516 SOD1 G93A RNS60 vs SOD1 G93A NS) and IL-1β ( p = 0.7033 SOD1 G93A RNS60 vs SOD1 G93A NS) the increase observed in C57BL/6-SOD1 G93A mice treated with NS was unchanged by RNS60 treatment. On the contrary, as regard the anti-inflammatory markers such as IL-4 ( p = 0.0033 SOD1 G93A RNS60 vs SOD1 G93A NS) and YM1 ( p = 0.2861 SOD1 G93A RNS60 vs SOD1 G93A NS), we observed a clear increase in RNS60 treated mice suggesting an anti-inflammatory response, even if the increase of YM1 did not reach statistical significance. Data are normalized to β-actin and expressed as the mean ± SEM fold change ratio between the two transgenic mice groups as percentage versus NTG ( n = 5 mice per group). All data were statistically analyzed using one-way ANOVA followed by post hoc Fisher’s LSD. * p
Figure Legend Snippet: RNS60 activates astrocytes and phagocytic microglia and increases anti-inflammatory molecules in C57BL/6-SOD1 G93A mice. a – c Representative images of ventral LSC hemisections stained with GFAP (green) at 20 weeks. Scale bar: 50 μm. d Quantification of GFAP immunofluorescence showed elevated astrocytosis in the LSC ventral horn of C57BL/6-SOD1 G93A mice at the symptomatic stage of the pathology as compared to NTG expressed as 100%. Treatment with RNS60 significantly increased reactive astrocytosis. Bars are mean ± SEM as a percentage versus NTG controls ( n = 3 animals for each group). e – g IBA1-stained (light blue) LSC hemisection from NTG, and SOD1 G93A mice treated with NS or RNS60. Scale bar: 50 μm. h Relative quantification showed higher level of IBA1 immunofluorescence in transgenic mice compared to NTG, without changes after the treatment with RNS60. Data are expressed as mean ± SEM as percentage versus NTG ( n = 3 animals for each group). i – k CD68 positive macrophagic microglia (red) was labeled in ventral horn of LSC. Scale bar: 50 μm. l Immunoreactivity quantification showed an up-regulation of CD68 in the LSC of C57BL/6-SOD1G93A mice compared to NTG. This effect was exacerbated by the treatment with RNS60. The bar graph represents mean ± SEM as a percentage versus NTG controls ( n = 3 mice per group). m – p Real-time PCR for pro-inflammatory (CCL2 and IL-1β) and anti-inflammatory (IL-4 and YM1) markers in the ventral portion of LSC of NTG mice expressed as 100% and transgenic mice treated with NS or RNS60. For CCL2 ( p = 0.7516 SOD1 G93A RNS60 vs SOD1 G93A NS) and IL-1β ( p = 0.7033 SOD1 G93A RNS60 vs SOD1 G93A NS) the increase observed in C57BL/6-SOD1 G93A mice treated with NS was unchanged by RNS60 treatment. On the contrary, as regard the anti-inflammatory markers such as IL-4 ( p = 0.0033 SOD1 G93A RNS60 vs SOD1 G93A NS) and YM1 ( p = 0.2861 SOD1 G93A RNS60 vs SOD1 G93A NS), we observed a clear increase in RNS60 treated mice suggesting an anti-inflammatory response, even if the increase of YM1 did not reach statistical significance. Data are normalized to β-actin and expressed as the mean ± SEM fold change ratio between the two transgenic mice groups as percentage versus NTG ( n = 5 mice per group). All data were statistically analyzed using one-way ANOVA followed by post hoc Fisher’s LSD. * p

Techniques Used: Mouse Assay, Staining, Immunofluorescence, Transgenic Assay, Labeling, Real-time Polymerase Chain Reaction

10) Product Images from "Interleukin-1 Mediates Neuroinflammatory Changes Associated With Diet-Induced Atherosclerosis"

Article Title: Interleukin-1 Mediates Neuroinflammatory Changes Associated With Diet-Induced Atherosclerosis

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.112.002006

Levels of microglial activation differed between ApoE −/− mice fed a Paigen or Western diet. Levels of microglial activation were assessed through the immunostaining of Iba1. Microglial activation was assessed throughout the striatum, and the location of images shown is indicated in the schematic diagram. Levels of microglial activation differed for each diet, but IL-1β neutralization had no effect. Error bars indicate standard error (n=7), scale bars 200 μm and 10 μm.
Figure Legend Snippet: Levels of microglial activation differed between ApoE −/− mice fed a Paigen or Western diet. Levels of microglial activation were assessed through the immunostaining of Iba1. Microglial activation was assessed throughout the striatum, and the location of images shown is indicated in the schematic diagram. Levels of microglial activation differed for each diet, but IL-1β neutralization had no effect. Error bars indicate standard error (n=7), scale bars 200 μm and 10 μm.

Techniques Used: Activation Assay, Mouse Assay, Western Blot, Immunostaining, Neutralization

Vascular activation, microglial activation, and leukocyte accumulation are reduced in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet. Activated microglia as identified by increased Iba1 immunopositivity, thickened processes, and irregular cell bodies were seen in ApoE −/− mice fed a Paigen diet. Microglial activation was significantly reduced, to control levels, in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (A). Vascular activation was assessed through the immunostaining of the adhesion molecule VCAM. Atherosclerotic ApoE −/− mice show increased vascular activation, which is significantly reduced in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (B). Leukocyte accumulation as shown by CD45 immunostaining was increased in ApoE −/− mice fed a Paigen diet. Leukocyte accumulation was significantly reduced, to control levels, in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (C). D, Quantification of Iba1-positive microglia. E, Quantification of VCAM-positive blood vessels. F, Quantification of CD45-positive leukocytes. The “vehicle” data in F were not normally distributed; therefore, in addition to one-way ANOVA ( P =0.017) followed by Bonferroni multiple-comparison post-test, nonparametric Kruskal-Wallis test ( P =0.0065) followed by Dunn multiple-comparison test also were performed. Post-hoc comparisons gave identical results. A through F: n=6–10. G, Voluntary wheel running actogram for ApoE −/− and ApoE −/− /IL-1R1 −/− mice (n=2–3). Error bars represent standard error, * P
Figure Legend Snippet: Vascular activation, microglial activation, and leukocyte accumulation are reduced in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet. Activated microglia as identified by increased Iba1 immunopositivity, thickened processes, and irregular cell bodies were seen in ApoE −/− mice fed a Paigen diet. Microglial activation was significantly reduced, to control levels, in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (A). Vascular activation was assessed through the immunostaining of the adhesion molecule VCAM. Atherosclerotic ApoE −/− mice show increased vascular activation, which is significantly reduced in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (B). Leukocyte accumulation as shown by CD45 immunostaining was increased in ApoE −/− mice fed a Paigen diet. Leukocyte accumulation was significantly reduced, to control levels, in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (C). D, Quantification of Iba1-positive microglia. E, Quantification of VCAM-positive blood vessels. F, Quantification of CD45-positive leukocytes. The “vehicle” data in F were not normally distributed; therefore, in addition to one-way ANOVA ( P =0.017) followed by Bonferroni multiple-comparison post-test, nonparametric Kruskal-Wallis test ( P =0.0065) followed by Dunn multiple-comparison test also were performed. Post-hoc comparisons gave identical results. A through F: n=6–10. G, Voluntary wheel running actogram for ApoE −/− and ApoE −/− /IL-1R1 −/− mice (n=2–3). Error bars represent standard error, * P

Techniques Used: Activation Assay, Mouse Assay, Immunostaining

11) Product Images from "Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury"

Article Title: Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury

Journal: ASN NEURO

doi: 10.1177/1759091416630220

Ephrin-B1 immunoreactivity was significantly upregulated in reactive astrocytes in the hippocampus following moderate CCI. (a–d) Fluorescent images show GFAP-positive astrocytes (GFAP, red in a and d, gray in c), Iba1-positive microglia (Iba1, blue in a), and ephrin-B1 immunoreactivity (ephrin-B1, green in a, gray in b) in the SR area of the CA1 hippocampus in control, 1, 3, and 7 dpi. (d) High magnification images show examples of ephrin-B1-positive astrocytes. Note that ephrin-B1-positive immunoreactivity is found in the dendrites of CA1 neurons and astrocytes in SR area of CA1 hippocampus. (e–g) Graphs show GFAP immunoreactivity per GFAP-positive astrocyte (e) or ephrin-B1 immunoreactivity per ephrin-B1-positive astrocyte (f) in control ( n = 682 cells, 9 images, 3 mice), 1 dpi ( n = 385 cells, 5 images, 3 mice), 3 dpi ( n = 732 cells, 11 images, 4 mice), and 7 dpi ( n = 1217 cells, 9 images, 3 mice) or post-sham ( n = 300–500 cells, 4–6 images, 3 mice). Note that ephrin-B1 immunoreactivity was not detected in all GFAP-positive cells. Error bars indicate SEM . Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc analysis (e,f, n = 3–4 mice, * p
Figure Legend Snippet: Ephrin-B1 immunoreactivity was significantly upregulated in reactive astrocytes in the hippocampus following moderate CCI. (a–d) Fluorescent images show GFAP-positive astrocytes (GFAP, red in a and d, gray in c), Iba1-positive microglia (Iba1, blue in a), and ephrin-B1 immunoreactivity (ephrin-B1, green in a, gray in b) in the SR area of the CA1 hippocampus in control, 1, 3, and 7 dpi. (d) High magnification images show examples of ephrin-B1-positive astrocytes. Note that ephrin-B1-positive immunoreactivity is found in the dendrites of CA1 neurons and astrocytes in SR area of CA1 hippocampus. (e–g) Graphs show GFAP immunoreactivity per GFAP-positive astrocyte (e) or ephrin-B1 immunoreactivity per ephrin-B1-positive astrocyte (f) in control ( n = 682 cells, 9 images, 3 mice), 1 dpi ( n = 385 cells, 5 images, 3 mice), 3 dpi ( n = 732 cells, 11 images, 4 mice), and 7 dpi ( n = 1217 cells, 9 images, 3 mice) or post-sham ( n = 300–500 cells, 4–6 images, 3 mice). Note that ephrin-B1 immunoreactivity was not detected in all GFAP-positive cells. Error bars indicate SEM . Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc analysis (e,f, n = 3–4 mice, * p

Techniques Used: Mouse Assay

12) Product Images from "Glycolic acid protects neurons against ischemia in vitro and in two animal models of stroke"

Article Title: Glycolic acid protects neurons against ischemia in vitro and in two animal models of stroke

Journal: bioRxiv

doi: 10.1101/2020.11.24.396051

GA mitigates the increase in the amount of astrocytes and microglia in large infarcts after MCAo. A) Fluorescence microscopy images showing the staining for microglia (IBA1, green) and astrocytes (GFAP, red) in a sham brain section (i), as well as a vehicle (ii) and GA brain section with large infarcts. iv) Same brain section as in (iii) indicating the outlines of the different areas in which Mean Fluorescence Intensity (MFI) was measured. B) i) MFI of IBA1 staining in the ischemic area showing no significant differences between groups (1-way ANOVA, p=0,1482) ii) When large infarcts ( > 18% of hemispheric volume) are analyzed separately, the MFI of IBA1 inside the infarct area is significantly increased in the vehicle group compared to sham (mean diff. =-11,31, p
Figure Legend Snippet: GA mitigates the increase in the amount of astrocytes and microglia in large infarcts after MCAo. A) Fluorescence microscopy images showing the staining for microglia (IBA1, green) and astrocytes (GFAP, red) in a sham brain section (i), as well as a vehicle (ii) and GA brain section with large infarcts. iv) Same brain section as in (iii) indicating the outlines of the different areas in which Mean Fluorescence Intensity (MFI) was measured. B) i) MFI of IBA1 staining in the ischemic area showing no significant differences between groups (1-way ANOVA, p=0,1482) ii) When large infarcts ( > 18% of hemispheric volume) are analyzed separately, the MFI of IBA1 inside the infarct area is significantly increased in the vehicle group compared to sham (mean diff. =-11,31, p

Techniques Used: Fluorescence, Microscopy, Staining

13) Product Images from "Microglia permit climbing fiber elimination by promoting GABAergic inhibition in the developing cerebellum"

Article Title: Microglia permit climbing fiber elimination by promoting GABAergic inhibition in the developing cerebellum

Journal: Nature Communications

doi: 10.1038/s41467-018-05100-z

CF synapse elimination after P10–P12 is impaired in Csf1r -cKO mice. a (upper) Representative traces of CF-EPSCs in control (left), Iba1 -KO (middle), and Csf1r -cKO (right) PCs in response to electrical stimulation with gradually increasing stimulus intensities; Vh = −10 mV. (lower) Frequency distribution histograms showing the number of discrete CF-EPSC steps in control (blue), Iba1 -KO (green), and Csf1r -cKO (red) mice aged P21–P40. PCs were evenly sampled from all lobules. Data are sampled from four control, four Iba1 -KO, and five Csf1r -cKO mice. CF synapse elimination was normal in Iba1 -KO mice ( p = 0.887, Mann–Whitney U test) but significantly impaired in Csf1r -cKO mice ( p = 0.007). b Frequency distribution histograms of the number of CFs in control (blue) and Csf1r -cKO (red) mice at P60–P80 ( p = 0.004). c–f Developmental course of CF synapse elimination. Frequency distribution histograms of the number of CFs at P6–P8 ( c , p = 0.207), P10–P12 ( d , p = 0.034), P13–P15 ( e , p = 0.028), and P16–P18 ( f , p = 0.030). Data were obtained from 4 to 5 control and 4 to 5 Csf1r -cKO mice at each postnatal period. Scale bars, 1 nA and 10 ms. Vh = −70 mV ( c ) and −10 mV ( b, d – f ). * p
Figure Legend Snippet: CF synapse elimination after P10–P12 is impaired in Csf1r -cKO mice. a (upper) Representative traces of CF-EPSCs in control (left), Iba1 -KO (middle), and Csf1r -cKO (right) PCs in response to electrical stimulation with gradually increasing stimulus intensities; Vh = −10 mV. (lower) Frequency distribution histograms showing the number of discrete CF-EPSC steps in control (blue), Iba1 -KO (green), and Csf1r -cKO (red) mice aged P21–P40. PCs were evenly sampled from all lobules. Data are sampled from four control, four Iba1 -KO, and five Csf1r -cKO mice. CF synapse elimination was normal in Iba1 -KO mice ( p = 0.887, Mann–Whitney U test) but significantly impaired in Csf1r -cKO mice ( p = 0.007). b Frequency distribution histograms of the number of CFs in control (blue) and Csf1r -cKO (red) mice at P60–P80 ( p = 0.004). c–f Developmental course of CF synapse elimination. Frequency distribution histograms of the number of CFs at P6–P8 ( c , p = 0.207), P10–P12 ( d , p = 0.034), P13–P15 ( e , p = 0.028), and P16–P18 ( f , p = 0.030). Data were obtained from 4 to 5 control and 4 to 5 Csf1r -cKO mice at each postnatal period. Scale bars, 1 nA and 10 ms. Vh = −70 mV ( c ) and −10 mV ( b, d – f ). * p

Techniques Used: Mouse Assay, MANN-WHITNEY, Mass Spectrometry

Double staining of CFs and microglia in the developing cerebellum. a Orthogonal fluorescence images of Iba1-positive microglia (green) and CFs anterogradely labeled with Alexa Fluor 568-dextran (magenta) in C57BL/6 mice. The xy -image is a single plane with its position represented as white lines in the xz and zy images. b Serial enlarged images (∆ z = 1 μm, from the left top to the right bottom) of a region surrounded by a dotted line in ( a ). Alexa Fluor 568-labeled CFs were very closely associated with Iba1-positive microglial processes but were not internalized in microglia (arrows, arrowheads, and double arrowheads). c – f Orthogonal fluorescence images of other microglia. g One of the rare examples in which an isolated CF fragment was internalized in Iba1-positive microglia. h Orthogonal fluorescence images similar to a but taken from C57BL/6 mice administrated 3-AP, harmaline and nicotinamide. i Serial enlarged images of a region surrounded by a dotted line in h . j , k Orthogonal fluorescence images of other 3-AP treated microglia. Fragments of Alexa Fluor 568-labeled CFs are included in Iba1-positive microglia (arrows and arrowheads in h – k ), indicating that the fluorescence of Alexa Fluor 568 can be detected after being engulfed by microglia. Scale bars, 10 μm
Figure Legend Snippet: Double staining of CFs and microglia in the developing cerebellum. a Orthogonal fluorescence images of Iba1-positive microglia (green) and CFs anterogradely labeled with Alexa Fluor 568-dextran (magenta) in C57BL/6 mice. The xy -image is a single plane with its position represented as white lines in the xz and zy images. b Serial enlarged images (∆ z = 1 μm, from the left top to the right bottom) of a region surrounded by a dotted line in ( a ). Alexa Fluor 568-labeled CFs were very closely associated with Iba1-positive microglial processes but were not internalized in microglia (arrows, arrowheads, and double arrowheads). c – f Orthogonal fluorescence images of other microglia. g One of the rare examples in which an isolated CF fragment was internalized in Iba1-positive microglia. h Orthogonal fluorescence images similar to a but taken from C57BL/6 mice administrated 3-AP, harmaline and nicotinamide. i Serial enlarged images of a region surrounded by a dotted line in h . j , k Orthogonal fluorescence images of other 3-AP treated microglia. Fragments of Alexa Fluor 568-labeled CFs are included in Iba1-positive microglia (arrows and arrowheads in h – k ), indicating that the fluorescence of Alexa Fluor 568 can be detected after being engulfed by microglia. Scale bars, 10 μm

Techniques Used: Double Staining, Fluorescence, Labeling, Mouse Assay, Isolation

Postnatal changes in microglial distribution in the cerebellum. a Immunofluorescent labeling for Iba1 (green) and calbindin staining (red) in lobules IV–V in the vermis of mouse cerebellum at P5, P8, P14, P21, and P60. EGL external granule cell layer, ML molecular layer, PCL Purkinje cell layer, IGL internal granule cell layer, WM white matter. Scale bars, 50 μm. b Frequency distribution histograms for the relative depth of microglia at P5 ( n = 5 mice), P8–P9 ( n = 5), P13–P14 ( n = 4), P20–P21 ( n = 4), and P60 ( n = 6). The depth of microglia was normalized to the total length of the gray matter and the white matter where each microglial cell was sampled. c Postnatal changes in densities of microglia in the WM (blue) and the gray matter (orange). Numbers of mice at individual postnatal days are same as in ( b ). d Postnatal changes in the total length of the ML, PCL, IGL, and WM. e Frequency distribution histograms of the relative depth of microglia in the gray matter except the EGL (the ML, the PCL, and the IGL) at P8–P9 (left, n = 5 mice) and P13–P14 (right, n = 4). Note that microglia tend to aggregate beneath the part of the PCL (the area between orange broken lines) and the edge beside the WM at P8–P9; * p
Figure Legend Snippet: Postnatal changes in microglial distribution in the cerebellum. a Immunofluorescent labeling for Iba1 (green) and calbindin staining (red) in lobules IV–V in the vermis of mouse cerebellum at P5, P8, P14, P21, and P60. EGL external granule cell layer, ML molecular layer, PCL Purkinje cell layer, IGL internal granule cell layer, WM white matter. Scale bars, 50 μm. b Frequency distribution histograms for the relative depth of microglia at P5 ( n = 5 mice), P8–P9 ( n = 5), P13–P14 ( n = 4), P20–P21 ( n = 4), and P60 ( n = 6). The depth of microglia was normalized to the total length of the gray matter and the white matter where each microglial cell was sampled. c Postnatal changes in densities of microglia in the WM (blue) and the gray matter (orange). Numbers of mice at individual postnatal days are same as in ( b ). d Postnatal changes in the total length of the ML, PCL, IGL, and WM. e Frequency distribution histograms of the relative depth of microglia in the gray matter except the EGL (the ML, the PCL, and the IGL) at P8–P9 (left, n = 5 mice) and P13–P14 (right, n = 4). Note that microglia tend to aggregate beneath the part of the PCL (the area between orange broken lines) and the edge beside the WM at P8–P9; * p

Techniques Used: Labeling, Staining, Mouse Assay

Effects of pharmacological deletion of microglia on CF synapse elimination. a–f Double immunolabeling for Iba1 (red) and calbindin (green) in liposomal clodronate ( a – c ) or control liposome ( d – f ) injected mice at P8. Asterisks in a and d indicate the injection sites of the liposomes in lobules VI–VII in the vermis. Regions surrounded by solid and dotted lines are magnified in b and e , and c and f , respectively. Iba1-labeled microglial cell numbers are significantly reduced in the clodronate-injected lobules VI–VII ( b ), but not in the neighboring lobules IV–V ( c ). g, h (left) Representative traces of CF-EPSCs recorded in mice treated with control (upper) or clodronate (lower) liposomes at P6 ( g ) or P11 ( h ). Electrophysiological recordings were conducted from PCs in the liposome-injected lobules at P21–P31. The holding potential (Vh) is −10 mV. Scale bars, 1 nA ( g ), 0.5 nA ( h ) and 10 ms. (right) Frequency distribution histograms showing the number of discrete CF-EPSC steps. Data were sampled from five control and five clodronate-treated mice in each of g and h . CF synapse elimination was significantly impaired in mice treated with clodronate at P6 ( g , p
Figure Legend Snippet: Effects of pharmacological deletion of microglia on CF synapse elimination. a–f Double immunolabeling for Iba1 (red) and calbindin (green) in liposomal clodronate ( a – c ) or control liposome ( d – f ) injected mice at P8. Asterisks in a and d indicate the injection sites of the liposomes in lobules VI–VII in the vermis. Regions surrounded by solid and dotted lines are magnified in b and e , and c and f , respectively. Iba1-labeled microglial cell numbers are significantly reduced in the clodronate-injected lobules VI–VII ( b ), but not in the neighboring lobules IV–V ( c ). g, h (left) Representative traces of CF-EPSCs recorded in mice treated with control (upper) or clodronate (lower) liposomes at P6 ( g ) or P11 ( h ). Electrophysiological recordings were conducted from PCs in the liposome-injected lobules at P21–P31. The holding potential (Vh) is −10 mV. Scale bars, 1 nA ( g ), 0.5 nA ( h ) and 10 ms. (right) Frequency distribution histograms showing the number of discrete CF-EPSC steps. Data were sampled from five control and five clodronate-treated mice in each of g and h . CF synapse elimination was significantly impaired in mice treated with clodronate at P6 ( g , p

Techniques Used: Immunolabeling, Injection, Mouse Assay, Labeling, Mass Spectrometry

Microglia-selective deletion of Csf1r results in significant loss of microglia in the developing cerebellum. a – l Immunostaining for CX3CR1 (red), Iba1 (green), and DAPI (blue) in control mice ( a – i ) and Csf1r -cKO mice ( j – l ) at P7 (left), P14 (middle), and P21 (right). Images are maximum z -projections of slices with thicknesses of 50 μm taken from lobules VI–V in the vermis. In Csf1r -cKO mice, CX3CR1-positive cells were almost absent at P7 ( j ), and sparse at P14 ( k ) and P21 ( l ). m , n Enlargements of the CX3CR1-positive cells surrounded by dashed squares in h and k , respectively. o Summary box plots showing postnatal changes of the density of CX3CR1-positive cells in the IGL and ML in control (blue) and Csf1r -cKO (red) mice. The density of CX3CR1-positive microglia was significantly lower in Csf1r -cKO mice than control mice at all postnatal days that we examined ( p
Figure Legend Snippet: Microglia-selective deletion of Csf1r results in significant loss of microglia in the developing cerebellum. a – l Immunostaining for CX3CR1 (red), Iba1 (green), and DAPI (blue) in control mice ( a – i ) and Csf1r -cKO mice ( j – l ) at P7 (left), P14 (middle), and P21 (right). Images are maximum z -projections of slices with thicknesses of 50 μm taken from lobules VI–V in the vermis. In Csf1r -cKO mice, CX3CR1-positive cells were almost absent at P7 ( j ), and sparse at P14 ( k ) and P21 ( l ). m , n Enlargements of the CX3CR1-positive cells surrounded by dashed squares in h and k , respectively. o Summary box plots showing postnatal changes of the density of CX3CR1-positive cells in the IGL and ML in control (blue) and Csf1r -cKO (red) mice. The density of CX3CR1-positive microglia was significantly lower in Csf1r -cKO mice than control mice at all postnatal days that we examined ( p

Techniques Used: Immunostaining, Mouse Assay

Generation of the Iba1-iCre mouse line. a Schema of Iba1 genomic DNA and the targeted genome. Open and filled boxes indicate noncoding and coding exons, respectively. Filled circles in the Iba1 iCre allele delineate the 5′ and 3′ termini of the targeting vector. The vector was constructed to insert an improved Cre recombinase gene ( iCre ), followed by an internal ribosome entry site ( IRES ) and a FLAG-tagged EGFP , in frame into the translational initiation site of the Iba1 gene. Blue arrows show primer positions for PCR genotyping. Blue bars indicate probes for Southern blot analysis. Two frt sequences (semicircles) are attached to remove the neomycin resistance gene (Neo). E, Eco RV; N, Nde I. b Southern blot analysis for genomic DNAs from wild-type (+/+) and targeted ( +/iCre ) ES cells. Left, Eco RV-digested genomic DNA hybridized with 5′ probe; middle and right, Nde I-digested DNA hybridized with Neo or 3′ probe, respectively. c Genomic PCR genotyping of Iba1 +/iCre intercrosses using primers F, R1 and R2. d Immunoblots of crude fractions from whole mouse brains with anti-Iba1 and actin antibodies. e – i Cre recombinase activity induced by Iba1-iCre in CAG-floxed STOP tdTomato reporter mice. No EGFP fluorescence was detected in the Iba1-iCre cerebellum. Immunostaining for Iba1 (green) and tdTomato fluorescence (red) in lobules IV–V in the vermis of Iba1 iCre/+ ; tdTomato ( e – g ) and control ( Iba1 +/+ ; tdTomato ) ( h , i ) cerebella at P30. All Iba1-positive cells expressed tdTomato, but there were several tdTomato-labeled processes with very weak or no Iba1 immunoreactivity (arrowheads in e – p ). j – n Immunostaining for cell markers (green) in Iba1 iCre/+ ; tdTomato mice at P30; tdTomato did not overlap with markers for astrocyte (GFAP, j ), oligodendrocyte (APC, k ), NG2 chondroitin sulfate proteoglycan ( l ), neurons (NeuN, m ) or inhibitory neurons (parvalbumin, n ). o – r Localization of Iba1 and tdTomato signals at P7 ( o ) or P14 ( p ). q , r Fractions of tdTomato-labeled cells relative to Iba1-positive cells ( q ) or Iba1-negative cells relative to tdTomato-labeled cells ( r ) ( n = 310 cells from two mice at P7, n = 191 cells from two mice at P14). A scale bar, 50 μm ( e – p ). Error bars, SEM
Figure Legend Snippet: Generation of the Iba1-iCre mouse line. a Schema of Iba1 genomic DNA and the targeted genome. Open and filled boxes indicate noncoding and coding exons, respectively. Filled circles in the Iba1 iCre allele delineate the 5′ and 3′ termini of the targeting vector. The vector was constructed to insert an improved Cre recombinase gene ( iCre ), followed by an internal ribosome entry site ( IRES ) and a FLAG-tagged EGFP , in frame into the translational initiation site of the Iba1 gene. Blue arrows show primer positions for PCR genotyping. Blue bars indicate probes for Southern blot analysis. Two frt sequences (semicircles) are attached to remove the neomycin resistance gene (Neo). E, Eco RV; N, Nde I. b Southern blot analysis for genomic DNAs from wild-type (+/+) and targeted ( +/iCre ) ES cells. Left, Eco RV-digested genomic DNA hybridized with 5′ probe; middle and right, Nde I-digested DNA hybridized with Neo or 3′ probe, respectively. c Genomic PCR genotyping of Iba1 +/iCre intercrosses using primers F, R1 and R2. d Immunoblots of crude fractions from whole mouse brains with anti-Iba1 and actin antibodies. e – i Cre recombinase activity induced by Iba1-iCre in CAG-floxed STOP tdTomato reporter mice. No EGFP fluorescence was detected in the Iba1-iCre cerebellum. Immunostaining for Iba1 (green) and tdTomato fluorescence (red) in lobules IV–V in the vermis of Iba1 iCre/+ ; tdTomato ( e – g ) and control ( Iba1 +/+ ; tdTomato ) ( h , i ) cerebella at P30. All Iba1-positive cells expressed tdTomato, but there were several tdTomato-labeled processes with very weak or no Iba1 immunoreactivity (arrowheads in e – p ). j – n Immunostaining for cell markers (green) in Iba1 iCre/+ ; tdTomato mice at P30; tdTomato did not overlap with markers for astrocyte (GFAP, j ), oligodendrocyte (APC, k ), NG2 chondroitin sulfate proteoglycan ( l ), neurons (NeuN, m ) or inhibitory neurons (parvalbumin, n ). o – r Localization of Iba1 and tdTomato signals at P7 ( o ) or P14 ( p ). q , r Fractions of tdTomato-labeled cells relative to Iba1-positive cells ( q ) or Iba1-negative cells relative to tdTomato-labeled cells ( r ) ( n = 310 cells from two mice at P7, n = 191 cells from two mice at P14). A scale bar, 50 μm ( e – p ). Error bars, SEM

Techniques Used: Plasmid Preparation, Construct, Polymerase Chain Reaction, Southern Blot, Western Blot, Activity Assay, Mouse Assay, Fluorescence, Immunostaining, Labeling

14) Product Images from "Lymphotoxin-alpha expression in the meninges causes lymphoid tissue formation and neurodegeneration"

Article Title: Lymphotoxin-alpha expression in the meninges causes lymphoid tissue formation and neurodegeneration

Journal: bioRxiv

doi: 10.1101/2021.04.27.441396

Overexpression of LTα in the subarachnoid space induces meningeal inflammation. (A) DAPI nuclear staining showing the accumulation of cells down the entire length of the sagittal sulcus in both IFA and MOG immunised animals at 28 and 90dpi. No accumulation of cells can be seen in GFP vector animals. Scale bar = 200µm (B) Immunostaining shows presence of CD4+ and CD8+ T-cells, CD79a+ B-cells and IBA1+ myeloid cells within the dense infiltrates. Scale bar =100µm. (C-D) Quantification of the numbers of CD4+ (C) and CD8+ (D) cells/mm2 down the length of the SAS in all animal groups. Data presented as mean ± SEM (n=3-4 per group). **** P
Figure Legend Snippet: Overexpression of LTα in the subarachnoid space induces meningeal inflammation. (A) DAPI nuclear staining showing the accumulation of cells down the entire length of the sagittal sulcus in both IFA and MOG immunised animals at 28 and 90dpi. No accumulation of cells can be seen in GFP vector animals. Scale bar = 200µm (B) Immunostaining shows presence of CD4+ and CD8+ T-cells, CD79a+ B-cells and IBA1+ myeloid cells within the dense infiltrates. Scale bar =100µm. (C-D) Quantification of the numbers of CD4+ (C) and CD8+ (D) cells/mm2 down the length of the SAS in all animal groups. Data presented as mean ± SEM (n=3-4 per group). **** P

Techniques Used: Over Expression, Staining, Immunofluorescence, Plasmid Preparation, Immunostaining

Subpial demyelination following injection of recombinant LTα+IFNγ into the subarachnoid space. (A-O) Immunofluorescence for myelin oligodendrocyte glycoprotein (MOG) and the microglial marker IBA1 in rmMOG immunised rats injected with PBS (A-C) or 1mg LTα and 75ng IFNγ recombinant cytokines culled at 3 (D-F) , 7 (G-I) , 14 (J-L) and 21 (M-O) days post-cytokine injection (dpi), demonstrating the degree of demyelination and microglial activation at various times points. Cortex and midline images are magnifications of the regions shown by the boxed areas in image A. Scale bars: A = 500µm, B and C = 50µm. (P-Q) Quantification of subpial demyelination at the injection site in layer I (P) and layers II-III (Q) , presented as the percentage area covered by MOG immunofluorescence (IF) (n=3-4 each group). Data is presented as the mean ± SEM. Statistics: 1-way analysis of variance with Tukey post-test. * P
Figure Legend Snippet: Subpial demyelination following injection of recombinant LTα+IFNγ into the subarachnoid space. (A-O) Immunofluorescence for myelin oligodendrocyte glycoprotein (MOG) and the microglial marker IBA1 in rmMOG immunised rats injected with PBS (A-C) or 1mg LTα and 75ng IFNγ recombinant cytokines culled at 3 (D-F) , 7 (G-I) , 14 (J-L) and 21 (M-O) days post-cytokine injection (dpi), demonstrating the degree of demyelination and microglial activation at various times points. Cortex and midline images are magnifications of the regions shown by the boxed areas in image A. Scale bars: A = 500µm, B and C = 50µm. (P-Q) Quantification of subpial demyelination at the injection site in layer I (P) and layers II-III (Q) , presented as the percentage area covered by MOG immunofluorescence (IF) (n=3-4 each group). Data is presented as the mean ± SEM. Statistics: 1-way analysis of variance with Tukey post-test. * P

Techniques Used: Injection, Recombinant, Immunofluorescence, Marker, Activation Assay

15) Product Images from "Early Proliferation Does Not Prevent the Loss of Oligodendrocyte Progenitor Cells during the Chronic Phase of Secondary Degeneration in a CNS White Matter Tract"

Article Title: Early Proliferation Does Not Prevent the Loss of Oligodendrocyte Progenitor Cells during the Chronic Phase of Secondary Degeneration in a CNS White Matter Tract

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065710

Proliferation of oligodendroglia subpopulations following partial ON transection. Oligodendroglia and other olig2+ glia were identified with antibodies to NG2 (A), olig2 (B) and Ki67 (C), or with CC1 (E), olig2 (F) and Ki67 (G). D: Cells indicated are Ki67+/NG2+/olig2+ ( > ) and Ki67−/NG2+/olig2+ ( > > ). H: Cells indicated are Ki67+/CC1+/olig2+ ( > ) and Ki67−/CC1+/olig2+ ( > > ). Proliferating Ki67+/IBA1+ cells (I, indicated by > ) and to a lesser extent Ki67+/GFAP+ cells (J) were observed after injury; representative examples at 3 days shown. K–P: Quantification of the mean density ± S.E of oligodendroglia and other olig2+ glia populations following partial transection. Densities of Ki67– cells are represented by black bars while densities of Ki67+ cells are represented by red bars and differences from control indicated by Δ(p≤0.05). Overall differences in total density (combined Ki67+ and Ki67– values) compared to control are indicated by *(p≤0.05). Q: Summary graph of Ki67+ mean densities of all oligodendroglia and other olig2+ glia subpopulations. Scale bar A–H: 20 µm, I–J: 10 µm.
Figure Legend Snippet: Proliferation of oligodendroglia subpopulations following partial ON transection. Oligodendroglia and other olig2+ glia were identified with antibodies to NG2 (A), olig2 (B) and Ki67 (C), or with CC1 (E), olig2 (F) and Ki67 (G). D: Cells indicated are Ki67+/NG2+/olig2+ ( > ) and Ki67−/NG2+/olig2+ ( > > ). H: Cells indicated are Ki67+/CC1+/olig2+ ( > ) and Ki67−/CC1+/olig2+ ( > > ). Proliferating Ki67+/IBA1+ cells (I, indicated by > ) and to a lesser extent Ki67+/GFAP+ cells (J) were observed after injury; representative examples at 3 days shown. K–P: Quantification of the mean density ± S.E of oligodendroglia and other olig2+ glia populations following partial transection. Densities of Ki67– cells are represented by black bars while densities of Ki67+ cells are represented by red bars and differences from control indicated by Δ(p≤0.05). Overall differences in total density (combined Ki67+ and Ki67– values) compared to control are indicated by *(p≤0.05). Q: Summary graph of Ki67+ mean densities of all oligodendroglia and other olig2+ glia subpopulations. Scale bar A–H: 20 µm, I–J: 10 µm.

Techniques Used:

16) Product Images from "The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination"

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

Journal: Pharmacological Research

doi: 10.1016/j.phrs.2020.104997

Agathisflavone modulates microglia-oligodendrocyte interactions. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Photomicrographs of IBA1 immunostaining (red) and SOX10-EGFP+ oligodendrocytes (green) showing oligodendrocytes-microglia contacts in the different treatment groups; scale bar 20 μm. (B) Diagram illustrating microglial processes contacting oligodendrocytes body (Pr-B), or apposition of microglial and oligodendrocyte cell bodies (B—B). (C) Grouped bar graph showing the number of microglial contacts per SOX10+ cells; data are expressed as the mean ± SEM (n = 6), * p
Figure Legend Snippet: Agathisflavone modulates microglia-oligodendrocyte interactions. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Photomicrographs of IBA1 immunostaining (red) and SOX10-EGFP+ oligodendrocytes (green) showing oligodendrocytes-microglia contacts in the different treatment groups; scale bar 20 μm. (B) Diagram illustrating microglial processes contacting oligodendrocytes body (Pr-B), or apposition of microglial and oligodendrocyte cell bodies (B—B). (C) Grouped bar graph showing the number of microglial contacts per SOX10+ cells; data are expressed as the mean ± SEM (n = 6), * p

Techniques Used: Mouse Assay, Immunostaining

Estrogen receptor (ER) activation is required for agathisflavone to inhibit microgliosis and promote remyelination. (A) Root mean square deviation (RMSD) values and rod representation of crystallographic ligand pose (lilac) and the best pose of this ligand generated by DOCK 6.8 (yellow) for each complex. Distances less than 2 Å between the calculated pose and the crystallographic pose indicates that the program was successful in reproducing the experimental data (B) Representation of interactions between agathisflavone (FAB) and retinoic and estrogen receptors; the captions are described in the figure. (C–G) Organotypic cerebellar slices from SOX10-EGFP animals were maintained for 7DIV, then exposed to LPC for 15–17 h, followed by 2 h pretreatment with the selective ER-α antagonist MPP dihydrochloride at 10 nM (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole-dihydrochloride), or the selective ER-β antagonist PHTPP at 1 μM (4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5- a ]pyrimidin-3-yl]phenol), which were kept together with 10 μM FAB for a further 2 DIV. (C) Oligodendrocytes were identified by the Sox10-EGFP reporter (green) and slices were immunolabeled for MBP (red), neurofilament (blue) and Iba-1 (yellow); scale bar: 20 μm. Bar graphs compare LPC and LPC + FAB 10 μM with the effects of the ER antagonists MPP and PHTPP on the NF + axon index (D), the percentage of MBP+/NF + myelinated axons (E), the number of Sox10-EGFP + oligodendrocytes (F) and the number of Iba1+ microglia (G); data are expressed as the mean ± SEM (n = 5); ‡ p
Figure Legend Snippet: Estrogen receptor (ER) activation is required for agathisflavone to inhibit microgliosis and promote remyelination. (A) Root mean square deviation (RMSD) values and rod representation of crystallographic ligand pose (lilac) and the best pose of this ligand generated by DOCK 6.8 (yellow) for each complex. Distances less than 2 Å between the calculated pose and the crystallographic pose indicates that the program was successful in reproducing the experimental data (B) Representation of interactions between agathisflavone (FAB) and retinoic and estrogen receptors; the captions are described in the figure. (C–G) Organotypic cerebellar slices from SOX10-EGFP animals were maintained for 7DIV, then exposed to LPC for 15–17 h, followed by 2 h pretreatment with the selective ER-α antagonist MPP dihydrochloride at 10 nM (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole-dihydrochloride), or the selective ER-β antagonist PHTPP at 1 μM (4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5- a ]pyrimidin-3-yl]phenol), which were kept together with 10 μM FAB for a further 2 DIV. (C) Oligodendrocytes were identified by the Sox10-EGFP reporter (green) and slices were immunolabeled for MBP (red), neurofilament (blue) and Iba-1 (yellow); scale bar: 20 μm. Bar graphs compare LPC and LPC + FAB 10 μM with the effects of the ER antagonists MPP and PHTPP on the NF + axon index (D), the percentage of MBP+/NF + myelinated axons (E), the number of Sox10-EGFP + oligodendrocytes (F) and the number of Iba1+ microglia (G); data are expressed as the mean ± SEM (n = 5); ‡ p

Techniques Used: Activation Assay, Generated, Immunolabeling

Agathisflavone modifies microglial activation state. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Microglial proliferation was analyzed by immunolabelling for IBA1 (yellow) and Ki67 (red), counterstained with the nuclear dye Hoechst (blue). (B, C) Bar graph showing the number of IBA1+ microglia (B) and the percentage of IBA1+/Ki67+ proliferating microglia (C); data are expressed as the mean ± SEM (n = 5–11) and tested for significance using One-way ANOVA followed by Tukey’s post-hoc test. (D) Photomicrographs and binary and skeletonized IBA + microglia illustrating morphological differences in the different treatment groups; scale bar 50 μm. (E, F, G) Individual values violin plots of microglial soma size per microglial cell (20 microglial cells/image were analyzed) (E) and violin graphs of process endpoints (F) and length (G) per microglial cell; data are expressed as the median ± IQR; * p
Figure Legend Snippet: Agathisflavone modifies microglial activation state. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Microglial proliferation was analyzed by immunolabelling for IBA1 (yellow) and Ki67 (red), counterstained with the nuclear dye Hoechst (blue). (B, C) Bar graph showing the number of IBA1+ microglia (B) and the percentage of IBA1+/Ki67+ proliferating microglia (C); data are expressed as the mean ± SEM (n = 5–11) and tested for significance using One-way ANOVA followed by Tukey’s post-hoc test. (D) Photomicrographs and binary and skeletonized IBA + microglia illustrating morphological differences in the different treatment groups; scale bar 50 μm. (E, F, G) Individual values violin plots of microglial soma size per microglial cell (20 microglial cells/image were analyzed) (E) and violin graphs of process endpoints (F) and length (G) per microglial cell; data are expressed as the median ± IQR; * p

Techniques Used: Activation Assay, Mouse Assay

17) Product Images from "Brain region-specific enhancement of remyelination and prevention of demyelination by the CSF1R kinase inhibitor BLZ945"

Article Title: Brain region-specific enhancement of remyelination and prevention of demyelination by the CSF1R kinase inhibitor BLZ945

Journal: Acta Neuropathologica Communications

doi: 10.1186/s40478-018-0510-8

Prophylactic treatment with BLZ945 1 week before and during 5-week cuprizone intoxication inhibited demyelination and reduced microglia in the corpus callosum but enhanced axonal pathology and myelin debris in the external capsule. a Representative overview pictures from histological stainings of Luxol Fast Blue (LFB) for the different treatment groups at week5 (see Fig. 5a for the experimental setup and groups), red arrows: corpus callosum, green arrows: external capsule. b Corresponding analysis of the optical density (OD) of Luxol fast blue (LFB) in the cc and ec. c Representative overview and higher magnification pictures from immunohistological stainings detecting Iba1-positive microglia for the different treatment groups at week5 (see Fig. 5a for the experimental setup and groups), red arrows: corpus callosum, green arrows: external capsule. d Corresponding quantitative analysis of the immunohistochemistry for Iba1-positive microglia numbers in the corpus callosum and external capsule. Values were normalized to those of control vehicle mice. Group sizes: for all treatment groups n = 4–5. Data are shown as means±SEM. Scale bars: 200 μm for the higher magnification. Statistics: Turkey’s multiple comparison test one-way ANOVA (*: p
Figure Legend Snippet: Prophylactic treatment with BLZ945 1 week before and during 5-week cuprizone intoxication inhibited demyelination and reduced microglia in the corpus callosum but enhanced axonal pathology and myelin debris in the external capsule. a Representative overview pictures from histological stainings of Luxol Fast Blue (LFB) for the different treatment groups at week5 (see Fig. 5a for the experimental setup and groups), red arrows: corpus callosum, green arrows: external capsule. b Corresponding analysis of the optical density (OD) of Luxol fast blue (LFB) in the cc and ec. c Representative overview and higher magnification pictures from immunohistological stainings detecting Iba1-positive microglia for the different treatment groups at week5 (see Fig. 5a for the experimental setup and groups), red arrows: corpus callosum, green arrows: external capsule. d Corresponding quantitative analysis of the immunohistochemistry for Iba1-positive microglia numbers in the corpus callosum and external capsule. Values were normalized to those of control vehicle mice. Group sizes: for all treatment groups n = 4–5. Data are shown as means±SEM. Scale bars: 200 μm for the higher magnification. Statistics: Turkey’s multiple comparison test one-way ANOVA (*: p

Techniques Used: Immunohistochemistry, Mouse Assay

A 2-week therapeutic treatment with BLZ945 after a 5-week cuprizone intoxication period reduced microglia numbers but enhanced astrocytes. a Representative pictures from immunohistological stainings detecting the microglia marker Iba1 and glial fibrillary acidic protein (GFAP) astrocytes in the cortex for the different treatment groups at week 7 (see Fig. 2a for the experimental setup and groups). b, c, d Corresponding quantitative analysis of the immunohistochemistry for Iba1-positive microglia numbers and GFAP-positive astrocyte stained area in the cortex, striatum and corpus callosum/external capsule. Values were normalized to those of control, vehicle-treated mice. Group sizes: For all treatments n = 7. Data are shown as means±SEM. Scale bars: 100 μm. Statistics: Turkey’s multiple comparison test one-way ANOVA (**: p
Figure Legend Snippet: A 2-week therapeutic treatment with BLZ945 after a 5-week cuprizone intoxication period reduced microglia numbers but enhanced astrocytes. a Representative pictures from immunohistological stainings detecting the microglia marker Iba1 and glial fibrillary acidic protein (GFAP) astrocytes in the cortex for the different treatment groups at week 7 (see Fig. 2a for the experimental setup and groups). b, c, d Corresponding quantitative analysis of the immunohistochemistry for Iba1-positive microglia numbers and GFAP-positive astrocyte stained area in the cortex, striatum and corpus callosum/external capsule. Values were normalized to those of control, vehicle-treated mice. Group sizes: For all treatments n = 7. Data are shown as means±SEM. Scale bars: 100 μm. Statistics: Turkey’s multiple comparison test one-way ANOVA (**: p

Techniques Used: Marker, Immunohistochemistry, Staining, Mouse Assay

18) Product Images from "Colocalized, Bidirectional Optogenetic Modulations in Freely Behaving Animals with a Wireless Dual-Color Optoelectronic Probe"

Article Title: Colocalized, Bidirectional Optogenetic Modulations in Freely Behaving Animals with a Wireless Dual-Color Optoelectronic Probe

Journal: bioRxiv

doi: 10.1101/2021.06.02.446749

Immunoreactivity results showing the biocompatibility of the micro-LED probe, in comparison with those obtained with a silica based optical fiber. (a) Representative confocal fluorescence images of horizontal brain slices, showing immunohistochemical staining of astrocytes (GFAP) and activated microglia (Iba1) for both the LED probe and the fiber, after 1 day and 21 days implantation. Green: GFAP; Red: Iba1; Blue: DAPI. (b) Percentages of GFAP and Iba1 cell populations among DAPI cells collected at a distance of 200 μm from the edge of implantation. The LED probe and the fiber show similar results of inflammatory glial responses occurring after implantation. ( n = 3 mice for each group).
Figure Legend Snippet: Immunoreactivity results showing the biocompatibility of the micro-LED probe, in comparison with those obtained with a silica based optical fiber. (a) Representative confocal fluorescence images of horizontal brain slices, showing immunohistochemical staining of astrocytes (GFAP) and activated microglia (Iba1) for both the LED probe and the fiber, after 1 day and 21 days implantation. Green: GFAP; Red: Iba1; Blue: DAPI. (b) Percentages of GFAP and Iba1 cell populations among DAPI cells collected at a distance of 200 μm from the edge of implantation. The LED probe and the fiber show similar results of inflammatory glial responses occurring after implantation. ( n = 3 mice for each group).

Techniques Used: Fluorescence, Immunohistochemistry, Staining, Mouse Assay

19) Product Images from "Ccr2 deletion dissociates cavity size and tau pathology after mild traumatic brain injury"

Article Title: Ccr2 deletion dissociates cavity size and tau pathology after mild traumatic brain injury

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-015-0443-0

Modulation of the inflammatory reaction at 3 days after LFPI in CCR2 and CX3CR1 transgenic mice. Control Ccr2 RFP /+ ; Cx3cr1 GFP /+ ( a ), Ccr2 RFP / RFP ( b ), and Cx3cr1 GFP / GFP ( c ) mice were subjected to surgery (craniotomy; Sham mice) or mild injury, and brains were collected 3 days later. Serial sections were stained for GFP ( a , b (Mild), and c ) or Iba1 ( b (Surgery)) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Inflammatory cells are mostly restricted to the site of injury. Scale bar on images with whole slices, 1 cm. The brightness on the images was adjusted to allow an easier distinction of the brain sections from the background; all sections in a series were adjusted to the same brightness level. Magnified images show a close up view of microglial and monocyte distribution around the cavity size. Note the reduced number of RFP + cells in Ccr2 RFP / RFP mice. Scale bar for magnified images, 100 μm
Figure Legend Snippet: Modulation of the inflammatory reaction at 3 days after LFPI in CCR2 and CX3CR1 transgenic mice. Control Ccr2 RFP /+ ; Cx3cr1 GFP /+ ( a ), Ccr2 RFP / RFP ( b ), and Cx3cr1 GFP / GFP ( c ) mice were subjected to surgery (craniotomy; Sham mice) or mild injury, and brains were collected 3 days later. Serial sections were stained for GFP ( a , b (Mild), and c ) or Iba1 ( b (Surgery)) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Inflammatory cells are mostly restricted to the site of injury. Scale bar on images with whole slices, 1 cm. The brightness on the images was adjusted to allow an easier distinction of the brain sections from the background; all sections in a series were adjusted to the same brightness level. Magnified images show a close up view of microglial and monocyte distribution around the cavity size. Note the reduced number of RFP + cells in Ccr2 RFP / RFP mice. Scale bar for magnified images, 100 μm

Techniques Used: Transgenic Assay, Mouse Assay, Staining, Derivative Assay

Quantification of cavity volume and inflammatory reaction after TBI. Serial sections were stained for GFP (or Iba1) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Whole slices, lesion cavity, and area of the slices occupied by monocytes (RFP + cells) or microglia (increased GFP or Iba1 immunoreactivity) were manually outlined to calculate their area; all sections were summed up to calculate volumes. Pathology was quantified as a percentage of the analyzed brain tissue (4 mm total thickness) that contained the lesion cavity ( a ), infiltrating monocytes ( b ), or increased microglial staining ( c ). Statistics: two-way ANOVA and Tukey’s post hoc test. Comparisons between groups are shown with horizontal lines ; vertical line in figure legend indicates main effect of genotype. * p
Figure Legend Snippet: Quantification of cavity volume and inflammatory reaction after TBI. Serial sections were stained for GFP (or Iba1) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Whole slices, lesion cavity, and area of the slices occupied by monocytes (RFP + cells) or microglia (increased GFP or Iba1 immunoreactivity) were manually outlined to calculate their area; all sections were summed up to calculate volumes. Pathology was quantified as a percentage of the analyzed brain tissue (4 mm total thickness) that contained the lesion cavity ( a ), infiltrating monocytes ( b ), or increased microglial staining ( c ). Statistics: two-way ANOVA and Tukey’s post hoc test. Comparisons between groups are shown with horizontal lines ; vertical line in figure legend indicates main effect of genotype. * p

Techniques Used: Staining, Derivative Assay

20) Product Images from "Peripheral Tumor Necrosis Factor-Alpha (TNF-α) Modulates Amyloid Pathology by Regulating Blood-Derived Immune Cells and Glial Response in the Brain of AD/TNF Transgenic Mice"

Article Title: Peripheral Tumor Necrosis Factor-Alpha (TNF-α) Modulates Amyloid Pathology by Regulating Blood-Derived Immune Cells and Glial Response in the Brain of AD/TNF Transgenic Mice

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2484-16.2017

5XFAD/Tg197 brains display increased Ly6C + monocytes and CD68 + phagocytic macrophages/microglia that are reduced upon peripheral huTNF-α inhibition by infliximab. Elevated Ly6C and CD68 immunoreactivity represents a phenotypic characteristic of Tg197 mice. A , B , Ly6C/DAPI ( A ) and Ly6C/Iba1 ( B ) fluorescent staining in sagittal brain sections of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. Increased Ly6C + patrolling monocytes along the blood vessels can be detected in Tg197 and 5XFAD/Tg197 brains compared with the C57BL/6 and 5XFAD, whereas peripheral infliximab treatment of 5XFAD/Tg197 mice reduces Ly6C + monocytes ( A , arrowheads). Double Ly6C/Iba1immunostaining revealed that activated microglial cells with elongated morphology are detected in close association with Ly6C + monocytes in the brain blood vasculature of Tg197 and 5XFAD/Tg197 mice ( B , arrowheads). Microglial activation along the blood vessels is decreased in infliximab-treated 5XFAD/Tg197 mice ( B , arrowheads). C , Immunofluorescent detection of CD68/TMEM119 in sagittal brain sections of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. Sections were also stained for amyloid plaques with Thioflavine S. Microglia-specific TMEM119 immunoreactivity is decreased in Tg197 and 5XFAD/Tg197 brains and partially restored upon peripheral infliximab treatment of 5XFAD/Tg197 mice, although the pattern of expression remains abnormal. Tg197 and 5XFAD/Tg197 brains show increased phagocytic CD68 + TMEM119 + microglia (arrows) and CD68 + TMEM119 − macrophages (arrowheads) that are significantly reduced upon peripheral infliximab treatment of 5XFAD/Tg197 mice. CD68 + TMEM119 − macrophages surrounding amyloid deposits are detected in 5XFAD/Tg197 brains compared with plaque-associated CD68 + TMEM119 + microglia in 5XFAD and infliximab-treated 5XFAD/Tg197 brains. Representative pictures of the cortex are shown for each group. Scale bars, 50 μm.
Figure Legend Snippet: 5XFAD/Tg197 brains display increased Ly6C + monocytes and CD68 + phagocytic macrophages/microglia that are reduced upon peripheral huTNF-α inhibition by infliximab. Elevated Ly6C and CD68 immunoreactivity represents a phenotypic characteristic of Tg197 mice. A , B , Ly6C/DAPI ( A ) and Ly6C/Iba1 ( B ) fluorescent staining in sagittal brain sections of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. Increased Ly6C + patrolling monocytes along the blood vessels can be detected in Tg197 and 5XFAD/Tg197 brains compared with the C57BL/6 and 5XFAD, whereas peripheral infliximab treatment of 5XFAD/Tg197 mice reduces Ly6C + monocytes ( A , arrowheads). Double Ly6C/Iba1immunostaining revealed that activated microglial cells with elongated morphology are detected in close association with Ly6C + monocytes in the brain blood vasculature of Tg197 and 5XFAD/Tg197 mice ( B , arrowheads). Microglial activation along the blood vessels is decreased in infliximab-treated 5XFAD/Tg197 mice ( B , arrowheads). C , Immunofluorescent detection of CD68/TMEM119 in sagittal brain sections of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. Sections were also stained for amyloid plaques with Thioflavine S. Microglia-specific TMEM119 immunoreactivity is decreased in Tg197 and 5XFAD/Tg197 brains and partially restored upon peripheral infliximab treatment of 5XFAD/Tg197 mice, although the pattern of expression remains abnormal. Tg197 and 5XFAD/Tg197 brains show increased phagocytic CD68 + TMEM119 + microglia (arrows) and CD68 + TMEM119 − macrophages (arrowheads) that are significantly reduced upon peripheral infliximab treatment of 5XFAD/Tg197 mice. CD68 + TMEM119 − macrophages surrounding amyloid deposits are detected in 5XFAD/Tg197 brains compared with plaque-associated CD68 + TMEM119 + microglia in 5XFAD and infliximab-treated 5XFAD/Tg197 brains. Representative pictures of the cortex are shown for each group. Scale bars, 50 μm.

Techniques Used: Inhibition, Mouse Assay, Staining, Activation Assay, Expressing

huTNF-α expression in 5XFAD/Tg197 mice causes robust glial activation that is significantly decreased upon peripheral infliximab treatment. A , C , Immunofluorescent detection of Iba1-positive microglia ( A ) and GFAP-positive astrocytes ( C ) in sagittal brain sections of 2.5- and 4-month-old 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. Sections were also stained for amyloid plaques with Thioflavine S. 5XFAD mice display focal gliosis, with periplaque activation of microglia and astrocytes, whereas 5XFAD/Tg197 mice show diffused gliosis, with dispersed pattern of glial activation. In infliximab-treated 5XFAD/Tg197 mice, although microglial and astrocytic activation appears similar to 5XFAD controls, the immunoreactivity pattern remains diffused. Representative pictures of the cortex of 2.5- and 4-month-old mice are shown for each group. Scale bars, 50 μm. B , D , Western blot analysis of Iba1 ( B ) and GFAP ( D ) brain protein levels in 2.5-month-old 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. 5XFAD/Tg197 mice show strongly increased levels of Iba1 and GFAP that are significantly reduced upon peripheral infliximab treatment. Quantitation was performed with densitometric analysis using ImageJ software. Data are mean ± SEM; n = 2 per group; experiment repeated 3 times using different samples. ** p
Figure Legend Snippet: huTNF-α expression in 5XFAD/Tg197 mice causes robust glial activation that is significantly decreased upon peripheral infliximab treatment. A , C , Immunofluorescent detection of Iba1-positive microglia ( A ) and GFAP-positive astrocytes ( C ) in sagittal brain sections of 2.5- and 4-month-old 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. Sections were also stained for amyloid plaques with Thioflavine S. 5XFAD mice display focal gliosis, with periplaque activation of microglia and astrocytes, whereas 5XFAD/Tg197 mice show diffused gliosis, with dispersed pattern of glial activation. In infliximab-treated 5XFAD/Tg197 mice, although microglial and astrocytic activation appears similar to 5XFAD controls, the immunoreactivity pattern remains diffused. Representative pictures of the cortex of 2.5- and 4-month-old mice are shown for each group. Scale bars, 50 μm. B , D , Western blot analysis of Iba1 ( B ) and GFAP ( D ) brain protein levels in 2.5-month-old 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. 5XFAD/Tg197 mice show strongly increased levels of Iba1 and GFAP that are significantly reduced upon peripheral infliximab treatment. Quantitation was performed with densitometric analysis using ImageJ software. Data are mean ± SEM; n = 2 per group; experiment repeated 3 times using different samples. ** p

Techniques Used: Expressing, Mouse Assay, Activation Assay, Staining, Western Blot, Quantitation Assay, Software

Tg197 mice display increased glial activation compared with C57BL/6 mice, by the age of 1 month. A–D , Immunofluorescent detection of Iba1-positive microglia ( A , C ) and GFAP-positive astrocytes ( B , D ) in sagittal brain sections of 2.5- and 1-month-old Tg197 and C57BL/6 control mice. Diffused patterns of glial activation observed in 5XFAD/Tg197 mice comprise a feature of the Tg197 phenotype that develops as early as 1 month of age, before the onset of amyloid plaque formation or infliximab treatment. Representative pictures of the cortex are shown for each group. Scale bars, 250 μm.
Figure Legend Snippet: Tg197 mice display increased glial activation compared with C57BL/6 mice, by the age of 1 month. A–D , Immunofluorescent detection of Iba1-positive microglia ( A , C ) and GFAP-positive astrocytes ( B , D ) in sagittal brain sections of 2.5- and 1-month-old Tg197 and C57BL/6 control mice. Diffused patterns of glial activation observed in 5XFAD/Tg197 mice comprise a feature of the Tg197 phenotype that develops as early as 1 month of age, before the onset of amyloid plaque formation or infliximab treatment. Representative pictures of the cortex are shown for each group. Scale bars, 250 μm.

Techniques Used: Mouse Assay, Activation Assay

5XFAD/Tg197 mice display increased CD206-positive meningeal and perivascular macrophages that are reduced upon peripheral huTNF-α inhibition by infliximab. Elevated CD206-positive macrophages represent a phenotypic characteristic of Tg197 mice, whereas the presence of the 5XFAD transgene confers an additive effect. A–C , Double immunofluorescence for CD206/Iba1 ( A ), CD206/CD31 ( B ), and CD206/α-SMA ( C ) in sagittal brain sections of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. Arrowheads indicate meningeal macrophages. Arrows indicate perivascular CD206-positive macrophages. Representative pictures of the cortex and cortical vessels are shown for each group. Scale bars: A , C , 25 μm; B , 50 μm. D , Western blot analysis of CD206 brain protein levels in 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice revealed an increase in Tg197 and 5XFAD/Tg197 mice compared with the C57BL/6 and 5XFAD. Notably, 5XFAD/Tg197 mice show significantly increased CD206 levels compared with the Tg197. Infliximab-treated 5XFAD/Tg197 mice display reduced CD206 levels compared with the 5XFAD/Tg197. Quantitation was performed with densitometric analysis using ImageJ software. Data are mean ± SEM; n = 2 mice per group; experiment repeated 3 times. * p
Figure Legend Snippet: 5XFAD/Tg197 mice display increased CD206-positive meningeal and perivascular macrophages that are reduced upon peripheral huTNF-α inhibition by infliximab. Elevated CD206-positive macrophages represent a phenotypic characteristic of Tg197 mice, whereas the presence of the 5XFAD transgene confers an additive effect. A–C , Double immunofluorescence for CD206/Iba1 ( A ), CD206/CD31 ( B ), and CD206/α-SMA ( C ) in sagittal brain sections of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. Arrowheads indicate meningeal macrophages. Arrows indicate perivascular CD206-positive macrophages. Representative pictures of the cortex and cortical vessels are shown for each group. Scale bars: A , C , 25 μm; B , 50 μm. D , Western blot analysis of CD206 brain protein levels in 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice revealed an increase in Tg197 and 5XFAD/Tg197 mice compared with the C57BL/6 and 5XFAD. Notably, 5XFAD/Tg197 mice show significantly increased CD206 levels compared with the Tg197. Infliximab-treated 5XFAD/Tg197 mice display reduced CD206 levels compared with the 5XFAD/Tg197. Quantitation was performed with densitometric analysis using ImageJ software. Data are mean ± SEM; n = 2 mice per group; experiment repeated 3 times. * p

Techniques Used: Mouse Assay, Inhibition, Immunofluorescence, Western Blot, Quantitation Assay, Software

5XFAD/Tg197 brains show increased CD45 high microglia and infiltrating leukocytes that are reduced upon peripheral huTNF-α inhibition by infliximab. Elevated CD45 high leukocytes comprise a feature of Tg197 mice, whereas the presence of the 5XFAD transgene further enhances leukocyte infiltration. A–C , Immunofluorescent detection of CD45 ( A ) and double-labeling for CD45/Iba1 ( B ) and CD45/CD31 ( C ) in sagittal brain sections of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. A , CD45 identifies both resting (CD45 low ) and activated (CD45 high ) microglia and CD45 high round-shaped leukocytes (arrowheads). B , CD45 high round-shaped leukocytes are Iba1-negative. C , Arrowheads indicate CD45 high leukocytes in the parenchyma. Arrows indicate CD45 high leukocytes in close association with blood vessels. Representative pictures of the cortex ( A , B ) and corpus callosum ( C ) are shown for each group. Scale bars: A , 100 μm; B , C , 50 μm. D , Quantitation of CD45 high leukocytes in the cortex of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice revealed an increase in Tg197 and 5XFAD/Tg197 mice compared with the C57BL/6 and 5XFAD. Importantly, 5XFAD/Tg197 mice show significantly increased CD45 high leukocytes compared with the Tg197. Infliximab-treated 5XFAD/Tg197 mice display reduced number of CD45 high leukocytes compared with the 5XFAD/Tg197. Analysis was performed using ImageJ software. Data are mean ± SEM; n = 5 per group. * p
Figure Legend Snippet: 5XFAD/Tg197 brains show increased CD45 high microglia and infiltrating leukocytes that are reduced upon peripheral huTNF-α inhibition by infliximab. Elevated CD45 high leukocytes comprise a feature of Tg197 mice, whereas the presence of the 5XFAD transgene further enhances leukocyte infiltration. A–C , Immunofluorescent detection of CD45 ( A ) and double-labeling for CD45/Iba1 ( B ) and CD45/CD31 ( C ) in sagittal brain sections of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice. A , CD45 identifies both resting (CD45 low ) and activated (CD45 high ) microglia and CD45 high round-shaped leukocytes (arrowheads). B , CD45 high round-shaped leukocytes are Iba1-negative. C , Arrowheads indicate CD45 high leukocytes in the parenchyma. Arrows indicate CD45 high leukocytes in close association with blood vessels. Representative pictures of the cortex ( A , B ) and corpus callosum ( C ) are shown for each group. Scale bars: A , 100 μm; B , C , 50 μm. D , Quantitation of CD45 high leukocytes in the cortex of 2.5-month-old C57BL/6, Tg197, 5XFAD, 5XFAD/Tg197, and infliximab-treated (+infl) 5XFAD/Tg197 mice revealed an increase in Tg197 and 5XFAD/Tg197 mice compared with the C57BL/6 and 5XFAD. Importantly, 5XFAD/Tg197 mice show significantly increased CD45 high leukocytes compared with the Tg197. Infliximab-treated 5XFAD/Tg197 mice display reduced number of CD45 high leukocytes compared with the 5XFAD/Tg197. Analysis was performed using ImageJ software. Data are mean ± SEM; n = 5 per group. * p

Techniques Used: Inhibition, Mouse Assay, Labeling, Quantitation Assay, Software

21) Product Images from "Repeated Mild Closed Head Injuries Induce Long-Term White Matter Pathology and Neuronal Loss That Are Correlated With Behavioral Deficits"

Article Title: Repeated Mild Closed Head Injuries Induce Long-Term White Matter Pathology and Neuronal Loss That Are Correlated With Behavioral Deficits

Journal: ASN NEURO

doi: 10.1177/1759091418781921

Nod- scid gamma (NSG) mice do not exhibit changes in behavioral function nor white matter atrophy following rmCHI. (a) No differences were observed at 1 MPI on time in open arm, or (b) total distance traveled in NSG mice receiving 1 or 5 hits at 5 m/s. (c) No differences were observed in the latency to find a hidden platform during the Morris water maze testing at 2 MPI. (d) Corpus callosum atrophy was not observed at 6 MPI in NSG mice receiving any level of injury. In contrast to the gliosis and microglial inflammation observed in C57Bl/6J mice, there appeared to be reduced staining for GFAP (d) and Iba1 (e) in NSG mice following rmCHI. NSG mice were treated and stained identical to those shown in Figures 8 and 9 , although not in parallel with the staining of C57Bl/6J mice. rmCHI = repeated mild closed head injury; DPI = days postinjury; MPI = months postinjury; GFAP = glial fibrillary acidic protein.
Figure Legend Snippet: Nod- scid gamma (NSG) mice do not exhibit changes in behavioral function nor white matter atrophy following rmCHI. (a) No differences were observed at 1 MPI on time in open arm, or (b) total distance traveled in NSG mice receiving 1 or 5 hits at 5 m/s. (c) No differences were observed in the latency to find a hidden platform during the Morris water maze testing at 2 MPI. (d) Corpus callosum atrophy was not observed at 6 MPI in NSG mice receiving any level of injury. In contrast to the gliosis and microglial inflammation observed in C57Bl/6J mice, there appeared to be reduced staining for GFAP (d) and Iba1 (e) in NSG mice following rmCHI. NSG mice were treated and stained identical to those shown in Figures 8 and 9 , although not in parallel with the staining of C57Bl/6J mice. rmCHI = repeated mild closed head injury; DPI = days postinjury; MPI = months postinjury; GFAP = glial fibrillary acidic protein.

Techniques Used: Mouse Assay, Staining

rmCHI induces chronic microglial inflammation at 2 MPI and 6 MPI in white matter tracts. (a, d) Baseline levels of Iba1 immunoreactivity in sham C57Bl/6J mice. 1-hit mice have similar Iba1 immunoreactivity at 2 MPI (b, b') and 6 MPI (e, e') to sham mice at 2 MPI (a, a') and at (d, d') 6 MPI. (c, c') Mice receiving 10 hits have prominent Iba1 immunoreactivity at 2 MPI and (f, f') at 6 MPI, particularly in the corpus callosum. Low magnification scale bar = 1 mm; high magnification scale bar = 250 μm. rmCHI = repeated mild closed head injury; MPI = months postinjury.
Figure Legend Snippet: rmCHI induces chronic microglial inflammation at 2 MPI and 6 MPI in white matter tracts. (a, d) Baseline levels of Iba1 immunoreactivity in sham C57Bl/6J mice. 1-hit mice have similar Iba1 immunoreactivity at 2 MPI (b, b') and 6 MPI (e, e') to sham mice at 2 MPI (a, a') and at (d, d') 6 MPI. (c, c') Mice receiving 10 hits have prominent Iba1 immunoreactivity at 2 MPI and (f, f') at 6 MPI, particularly in the corpus callosum. Low magnification scale bar = 1 mm; high magnification scale bar = 250 μm. rmCHI = repeated mild closed head injury; MPI = months postinjury.

Techniques Used: Mouse Assay

22) Product Images from "Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice"

Article Title: Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice

Journal: eLife

doi: 10.7554/eLife.18128

SCs, not macrophages, produce phagosomes in adult mouse utricles under normal conditions. ( A,B ) Confocal projection images of the utricular macula from 7-week-old Plp1-CreER T2 :ROSA26 tdTomato mice showing the numbers and distribution of tdTomato-positive SCs (magenta) in the utricular sensory epithelium of mice that received no tamoxifen ( A ) or mice that received tamoxifen ( B ). S, striola; SE, sensory epithelium; TE, transitional epithelium. See Figure 4—source data 1 for quantification of tdTomato-positive SCs in extrastriolar and striolar regions. ( C–E’ ) Higher magnification optical sections of a Plp1-CreER T2 :ROSA26 tdTomato mouse utricular macula at one week post tamoxifen showing overlap between tdTomato-labeled SC cytoplasm (magenta) and a F-actin-rich phagosome (green, arrows). ( C ) Xy view with double lines indicating where cross-sectional images were created in D-E’ . ( D,D’ ) Yz view of same area in C .( E,E’ ) Xz view of same area in C . Very bright green labeling in D and E is F-actin in the stereocilia bundles of HCs. ( F,G ) Confocal optical sections of an adult Swiss Webster utricle. ( F ) Two F-actin-rich phagosomes (green, arrows) in the SC nuclear layer (DAPI, blue) did not co-label for antibodies to IBA1, a macrophage/monocyte lineage marker (magenta). ( F’ ) An IBA1-positive cell (magenta, arrowhead) resided in the connective tissue under the phagosomes shown in F . ( G ) Xz view of the field shown in F,F’ . The white dotted line indicates the border between sensory epithelium (SE) and connective tissue (CT). Arrows and arrowhead indicate the same cells shown in F,F’ . ( H ) Confocal projection image of the utricular macula from an adult Lfng-eGFP mouse showing eGFP-positive SCs (green) in the sensory epithelium (SE). S, Striola. ( I–I”’ ) Confocal optical sections of the same field from the extrastriolar region of a Lfng-eGFP utricle, with eGFP in green and F-actin in blue. ( I ) Xz view through the SE and CT of the utricle. ( I’,I” ) Xy views of the SC layer in the SE. ( I”’ ) Xy view of the CT. The dotted line in I is the approximate location of the basal lamina, between the SE and the CT. Two phagosomes (arrowheads, F-actin, blue) are flanked by eGFP-positive SCs (green) and are also co-labeled with eGFP (green); co-labeled phagosomes appear cyan. One phagosome (arrow in I’,I” ) is eGFP-negative and not flanked by eGFP-positive SCs. The double lines in I’–I”’ indicate the position of the xz view shown in I . Scale bar in A is 100 µm and applies to A , B , H . Scale bar in C is 5 µm and applies to C–G . Scale bar in I is 5 µm and applies to I–I”’ . DOI: http://dx.doi.org/10.7554/eLife.18128.010 10.7554/eLife.18128.011 Quantification of the percentage of SCs labeled with tdTomato in Plp1-CreER T2 :ROSA26 tdTomato utricles. Mean percentage {(one standard deviation, SD); [95% confidence interval, CI]} of tdTomato-labeled SCs in Plp1-CreER T2 :ROSA26 tdTomato mice given tamoxifen at 6 weeks (wks) of age and sacrificed 1 or 15 weeks post tamoxifen (7 or 21 weeks of age, respectively). SCs were sampled in two striolar regions and 6 extrastriolar regions in each utricle (see Materials and methods). n = 3 mice at both timepoints. DOI: http://dx.doi.org/10.7554/eLife.18128.011
Figure Legend Snippet: SCs, not macrophages, produce phagosomes in adult mouse utricles under normal conditions. ( A,B ) Confocal projection images of the utricular macula from 7-week-old Plp1-CreER T2 :ROSA26 tdTomato mice showing the numbers and distribution of tdTomato-positive SCs (magenta) in the utricular sensory epithelium of mice that received no tamoxifen ( A ) or mice that received tamoxifen ( B ). S, striola; SE, sensory epithelium; TE, transitional epithelium. See Figure 4—source data 1 for quantification of tdTomato-positive SCs in extrastriolar and striolar regions. ( C–E’ ) Higher magnification optical sections of a Plp1-CreER T2 :ROSA26 tdTomato mouse utricular macula at one week post tamoxifen showing overlap between tdTomato-labeled SC cytoplasm (magenta) and a F-actin-rich phagosome (green, arrows). ( C ) Xy view with double lines indicating where cross-sectional images were created in D-E’ . ( D,D’ ) Yz view of same area in C .( E,E’ ) Xz view of same area in C . Very bright green labeling in D and E is F-actin in the stereocilia bundles of HCs. ( F,G ) Confocal optical sections of an adult Swiss Webster utricle. ( F ) Two F-actin-rich phagosomes (green, arrows) in the SC nuclear layer (DAPI, blue) did not co-label for antibodies to IBA1, a macrophage/monocyte lineage marker (magenta). ( F’ ) An IBA1-positive cell (magenta, arrowhead) resided in the connective tissue under the phagosomes shown in F . ( G ) Xz view of the field shown in F,F’ . The white dotted line indicates the border between sensory epithelium (SE) and connective tissue (CT). Arrows and arrowhead indicate the same cells shown in F,F’ . ( H ) Confocal projection image of the utricular macula from an adult Lfng-eGFP mouse showing eGFP-positive SCs (green) in the sensory epithelium (SE). S, Striola. ( I–I”’ ) Confocal optical sections of the same field from the extrastriolar region of a Lfng-eGFP utricle, with eGFP in green and F-actin in blue. ( I ) Xz view through the SE and CT of the utricle. ( I’,I” ) Xy views of the SC layer in the SE. ( I”’ ) Xy view of the CT. The dotted line in I is the approximate location of the basal lamina, between the SE and the CT. Two phagosomes (arrowheads, F-actin, blue) are flanked by eGFP-positive SCs (green) and are also co-labeled with eGFP (green); co-labeled phagosomes appear cyan. One phagosome (arrow in I’,I” ) is eGFP-negative and not flanked by eGFP-positive SCs. The double lines in I’–I”’ indicate the position of the xz view shown in I . Scale bar in A is 100 µm and applies to A , B , H . Scale bar in C is 5 µm and applies to C–G . Scale bar in I is 5 µm and applies to I–I”’ . DOI: http://dx.doi.org/10.7554/eLife.18128.010 10.7554/eLife.18128.011 Quantification of the percentage of SCs labeled with tdTomato in Plp1-CreER T2 :ROSA26 tdTomato utricles. Mean percentage {(one standard deviation, SD); [95% confidence interval, CI]} of tdTomato-labeled SCs in Plp1-CreER T2 :ROSA26 tdTomato mice given tamoxifen at 6 weeks (wks) of age and sacrificed 1 or 15 weeks post tamoxifen (7 or 21 weeks of age, respectively). SCs were sampled in two striolar regions and 6 extrastriolar regions in each utricle (see Materials and methods). n = 3 mice at both timepoints. DOI: http://dx.doi.org/10.7554/eLife.18128.011

Techniques Used: Mouse Assay, Labeling, Marker, Standard Deviation

23) Product Images from "Microtubule-Associated Protein 1 Light Chain 3B, (LC3B) Is Necessary to Maintain Lipid-Mediated Homeostasis in the Retinal Pigment Epithelium"

Article Title: Microtubule-Associated Protein 1 Light Chain 3B, (LC3B) Is Necessary to Maintain Lipid-Mediated Homeostasis in the Retinal Pigment Epithelium

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00351

Loss of LC3B contributes to pro-inflammatory environment. (A) Immuno-staining of WT and LC3B -/- retinal sections with Iba1 showing numerous Iba1 positive cells (red, marked by dotted oval) in the L3CB -/- retina on the apical side of RPE, nuclei (blue); age 24 months. (B) Higher mag images showing the Iba1 positive cells (red) near the apical RPE, counterstained with phalloidin (green) to label the actin cytoskeleton; age ∼30 months. (C) Number of Iba1 positive cells per field in the superior and inferior RPE. Values are mean ± SEM from 4 individual mice (∼18 months), ∗∗ p
Figure Legend Snippet: Loss of LC3B contributes to pro-inflammatory environment. (A) Immuno-staining of WT and LC3B -/- retinal sections with Iba1 showing numerous Iba1 positive cells (red, marked by dotted oval) in the L3CB -/- retina on the apical side of RPE, nuclei (blue); age 24 months. (B) Higher mag images showing the Iba1 positive cells (red) near the apical RPE, counterstained with phalloidin (green) to label the actin cytoskeleton; age ∼30 months. (C) Number of Iba1 positive cells per field in the superior and inferior RPE. Values are mean ± SEM from 4 individual mice (∼18 months), ∗∗ p

Techniques Used: Immunostaining, Mouse Assay

24) Product Images from "Modified behavioural tests to detect white matter injury- induced motor deficits after intracerebral haemorrhage in mice"

Article Title: Modified behavioural tests to detect white matter injury- induced motor deficits after intracerebral haemorrhage in mice

Journal: Scientific Reports

doi: 10.1038/s41598-019-53263-6

Glia cell activation and myelin damage were assessed from day 1 to day 7 after ICH. ( A ) The schematic diagrams show the four areas (white squares) for immunofluorescence photography in the perihaematomal region, and the white star indicates the haematoma. Iba1, GFAP-positive cells and MBP in brain sections were identified on days 1, 3 and 7 after ICH. Immunoreactivity of Iba1 (ionized calcium-binding adaptor molecule 1) and GFAP (glial fibrillary acidic protein; astrocyte marker) is shown in green, MBP (myelin basic protein) is shown in red, and NF-200 (neurofilament-200) is shown in green. Nuclei were stained with DAPI (blue). Scale bars: 50 μm (inset column). ( B ) Intensity of Iba-1, GFAP and MBP staining and diameter of NF-200 were quantified; n = 3 mice and 6 pictures per time point. ( C ) TEM shows the myelin sheath around the haematoma on day 3 after ICH. ( D ) G-ratio of different groups (n = 60, 3 mice). ( E ) Diameter of axons (n = 6 slices, 3 mice). Values are shown as the mean ± S.D., *P
Figure Legend Snippet: Glia cell activation and myelin damage were assessed from day 1 to day 7 after ICH. ( A ) The schematic diagrams show the four areas (white squares) for immunofluorescence photography in the perihaematomal region, and the white star indicates the haematoma. Iba1, GFAP-positive cells and MBP in brain sections were identified on days 1, 3 and 7 after ICH. Immunoreactivity of Iba1 (ionized calcium-binding adaptor molecule 1) and GFAP (glial fibrillary acidic protein; astrocyte marker) is shown in green, MBP (myelin basic protein) is shown in red, and NF-200 (neurofilament-200) is shown in green. Nuclei were stained with DAPI (blue). Scale bars: 50 μm (inset column). ( B ) Intensity of Iba-1, GFAP and MBP staining and diameter of NF-200 were quantified; n = 3 mice and 6 pictures per time point. ( C ) TEM shows the myelin sheath around the haematoma on day 3 after ICH. ( D ) G-ratio of different groups (n = 60, 3 mice). ( E ) Diameter of axons (n = 6 slices, 3 mice). Values are shown as the mean ± S.D., *P

Techniques Used: Activation Assay, Immunofluorescence, Binding Assay, Marker, Staining, Mouse Assay, Transmission Electron Microscopy

25) Product Images from "Attenuated Inflammatory Response in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Knock-Out Mice following Stroke"

Article Title: Attenuated Inflammatory Response in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Knock-Out Mice following Stroke

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052982

Decreased microglial activation in TREM2-KO mice after stroke. A. Post-ischemic Iba1 positive cells (green) displayed the typical activated amoeboid phenotype in the glial scar and infarct core at 7 d and 28 d in WT mice, whereas they remained in a ramified phenotype in TREM2-KO mice. Nuclei of cells counterstained with DAPI (for a better distinction the color was changed to red). Scale bar 20 µm. B. Fewer Iba1 positive activated microglial cells were revealed in the glial scar of TREM2-KO mice (KO) at 7 d following stroke compared to littermate controls (WT). Bars represent mean ± s.e.m., n = 5/6 each, WT ipsi vs. KO ipsi ***p≤0.001. C. Gene transcripts for Iba1 and CD68 were determined at 12 h, 7 d and 28 d after stroke in TREM2-KO mice (KO) and littermate controls (WT). Attenuated gene transcription of Iba1 and CD68 was observed 7 d after stroke in TREM2-KO (KO) mice compared to control (WT) mice. Bars represent mean ± s.e.m. (mRNA copies per 1,000 Gapdh), n = 5/6 each, WT ipsi vs. KO ipsi *p≤0.05 (Iba1), + p≤0.05 (CD68).
Figure Legend Snippet: Decreased microglial activation in TREM2-KO mice after stroke. A. Post-ischemic Iba1 positive cells (green) displayed the typical activated amoeboid phenotype in the glial scar and infarct core at 7 d and 28 d in WT mice, whereas they remained in a ramified phenotype in TREM2-KO mice. Nuclei of cells counterstained with DAPI (for a better distinction the color was changed to red). Scale bar 20 µm. B. Fewer Iba1 positive activated microglial cells were revealed in the glial scar of TREM2-KO mice (KO) at 7 d following stroke compared to littermate controls (WT). Bars represent mean ± s.e.m., n = 5/6 each, WT ipsi vs. KO ipsi ***p≤0.001. C. Gene transcripts for Iba1 and CD68 were determined at 12 h, 7 d and 28 d after stroke in TREM2-KO mice (KO) and littermate controls (WT). Attenuated gene transcription of Iba1 and CD68 was observed 7 d after stroke in TREM2-KO (KO) mice compared to control (WT) mice. Bars represent mean ± s.e.m. (mRNA copies per 1,000 Gapdh), n = 5/6 each, WT ipsi vs. KO ipsi *p≤0.05 (Iba1), + p≤0.05 (CD68).

Techniques Used: Activation Assay, Mouse Assay

26) Product Images from "Neuroprotective function for ramified microglia in hippocampal excitotoxicity"

Article Title: Neuroprotective function for ramified microglia in hippocampal excitotoxicity

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-27

Microglial activation coincides with selective neuronal vulnerability towards excitotoxicity . Confocal images of microglia in control ( A - D ), 10 μM ( E - H ) and 15 μM ( I - L ) NMDA-treated slice cultures, as determined by Iba1-immuno-histochemistry. After 6 days in culture ( A ), microglia were evenly distributed throughout the slice cultures and displayed a typical ramified morphology ( B :CA1, C :CA3, D :DG). At 10 μM NMDA ( E ), changes in the CA1 region were clearly visible as numerous microglia accumulated at the site of injury ( E , arrow). Morphologically, these microglia displayed an
Figure Legend Snippet: Microglial activation coincides with selective neuronal vulnerability towards excitotoxicity . Confocal images of microglia in control ( A - D ), 10 μM ( E - H ) and 15 μM ( I - L ) NMDA-treated slice cultures, as determined by Iba1-immuno-histochemistry. After 6 days in culture ( A ), microglia were evenly distributed throughout the slice cultures and displayed a typical ramified morphology ( B :CA1, C :CA3, D :DG). At 10 μM NMDA ( E ), changes in the CA1 region were clearly visible as numerous microglia accumulated at the site of injury ( E , arrow). Morphologically, these microglia displayed an "activated" phenotype ( F , CA1) with enlarged somata and loss of secondary and tertiary branching. In contrast, accumulation of microglia did not occur in the CA3 ( G ) and DG ( H ) and these cells retained their ramified phenotype. At 15 μM NMDA, pronounced accumulation of morphologically activated microglia ( I , arrows) was observed in both CA1 ( J ) and CA3 ( K ). In contrast, microglia in the DG ( L ) showed only mild activation and accumulation of microglia was minimal in this region. Scale bars indicate 300 μM (overviews) and 25 μM (magnifications). Confocal images were gray-scaled and inversed.

Techniques Used: Activation Assay, Immunohistochemistry

Replenishment of microglia-depleted slice cultures with primary mouse microglia reduces excitotoxicity-induced neuronal cell death . After 9 days in vitro , cultured primary mouse microglia were carefully pipetted onto depleted slice cultures at a density of 400 cells per slice culture. 12 days later, slice cultures were immuno-stained for NeuN (grey) and Iba1 (yellow) revealing that exogenously applied microglia showed equal distribution and a ramified morphology ( A , D ) and were integrated into the tissue ( B ). The cells (yellow arrows) had distributed themselves throughout the total depth of the slice cultures as examined by confocal microscopy ( B : orthoview of a z-stack). 3D reconstructions of microglia filaments, created by IMARIS filament tracer software from Iba1 fluorescently stained cells in z-stacks of slice cultures, were used to analyse the morphology of endogenous microglia and replenished primary mouse microglia. Figure C and D show examples for reconstructions of endogenous microglia (endo microglia) and replenished primary microglia (primary microglia), respectively. The starting point of the filaments was set at the cell soma (blue). Analysis of the morphologic parameters total dendritic length ( E ) and number of branch points ( F ) revealed significantly shorter dendritic length and less branching points in replenished primary microglia compared to endogenous microglia (*** p
Figure Legend Snippet: Replenishment of microglia-depleted slice cultures with primary mouse microglia reduces excitotoxicity-induced neuronal cell death . After 9 days in vitro , cultured primary mouse microglia were carefully pipetted onto depleted slice cultures at a density of 400 cells per slice culture. 12 days later, slice cultures were immuno-stained for NeuN (grey) and Iba1 (yellow) revealing that exogenously applied microglia showed equal distribution and a ramified morphology ( A , D ) and were integrated into the tissue ( B ). The cells (yellow arrows) had distributed themselves throughout the total depth of the slice cultures as examined by confocal microscopy ( B : orthoview of a z-stack). 3D reconstructions of microglia filaments, created by IMARIS filament tracer software from Iba1 fluorescently stained cells in z-stacks of slice cultures, were used to analyse the morphology of endogenous microglia and replenished primary mouse microglia. Figure C and D show examples for reconstructions of endogenous microglia (endo microglia) and replenished primary microglia (primary microglia), respectively. The starting point of the filaments was set at the cell soma (blue). Analysis of the morphologic parameters total dendritic length ( E ) and number of branch points ( F ) revealed significantly shorter dendritic length and less branching points in replenished primary microglia compared to endogenous microglia (*** p

Techniques Used: In Vitro, Cell Culture, Staining, Confocal Microscopy, Software

27) Product Images from "iPSC-derived human microglia-like cells to study neurological diseases"

Article Title: iPSC-derived human microglia-like cells to study neurological diseases

Journal: Neuron

doi: 10.1016/j.neuron.2017.03.042

iMGLs transplanted into the brains of either wild-type or AD transplant competent mice are like brain microglia Within the brains of xenotransplantation compatible mice, transplanted iMGLs are ramified and interact with the neuronal environment. ( A–L ) After two months in vivo , iMGLs transplanted into mice display long-term viability with highly arborized processes resembling endogenous microglia found in the brain. ( A ) Transplanted iMGLs, labeled with P2ry12 (green; HPA HPA014518, Sigma) and human nuclei (ku80, red), exhibit long-term viability in mice. ( B–D ) At higher magnification, P2ry12 is highly expressed in iMGL arborized processes, both suggestive of homeostatic microglia surveying the brain environment. ( E–H ], and human cytoplasm maker SC121 (hCyto, red). ( I–L ) At higher magnification, representative iMGLs express P2ry12 (green), hCyto (red), and Iba1 (blue; ab5076, Abcam). ( M–P ) interact with and phagocytose amyloid plaques (white). ( I–J ) Transplanted iMGLs extend projections and migrate to plaques. iMGLs fully encompass amyloid plaques ( O ) and begin to phagocytose amyloid ( P ). Scale bars; ( A,E,N ) = 30 μm, ( B–D, F–H, I–L, O,P ) = 10 μm, ( M .
Figure Legend Snippet: iMGLs transplanted into the brains of either wild-type or AD transplant competent mice are like brain microglia Within the brains of xenotransplantation compatible mice, transplanted iMGLs are ramified and interact with the neuronal environment. ( A–L ) After two months in vivo , iMGLs transplanted into mice display long-term viability with highly arborized processes resembling endogenous microglia found in the brain. ( A ) Transplanted iMGLs, labeled with P2ry12 (green; HPA HPA014518, Sigma) and human nuclei (ku80, red), exhibit long-term viability in mice. ( B–D ) At higher magnification, P2ry12 is highly expressed in iMGL arborized processes, both suggestive of homeostatic microglia surveying the brain environment. ( E–H ], and human cytoplasm maker SC121 (hCyto, red). ( I–L ) At higher magnification, representative iMGLs express P2ry12 (green), hCyto (red), and Iba1 (blue; ab5076, Abcam). ( M–P ) interact with and phagocytose amyloid plaques (white). ( I–J ) Transplanted iMGLs extend projections and migrate to plaques. iMGLs fully encompass amyloid plaques ( O ) and begin to phagocytose amyloid ( P ). Scale bars; ( A,E,N ) = 30 μm, ( B–D, F–H, I–L, O,P ) = 10 μm, ( M .

Techniques Used: Mouse Assay, In Vivo, Labeling

28) Product Images from "One bout of neonatal inflammation impairs adult respiratory motor plasticity in male and female rats"

Article Title: One bout of neonatal inflammation impairs adult respiratory motor plasticity in male and female rats

Journal: eLife

doi: 10.7554/eLife.45399

Neonatal inflammation does not alter GFAP or IBA1 immunofluorescence in adult preBötzinger Complex or ventral cervical spinal cords. After neonatal LPS (1 mg/kg, i.p., (P4), representative confocal images (40x) from adult preBötC ( A and B ) and cervical spinal cords ( C and D ) displayed no qualitative differences in immunoreactivity for GFAP (green, astrocytes) or IBA1 (green, microglia) in males (left panels) or females (right panels). PreBötC neurons are labeled with antibodies for NK1R (red, A and B ) and motor neurons are labeled with antibodies for ChAT (red, C and D ). Neonatal inflammation did not significantly change mean fluorescent intensity of either GFAP or IBA1 in the preBötC ( E ) or cervical spinal cord ( F ), suggesting no lasting differences in astrocytes or microglia after neonatal inflammation. Scale bars: 50 µm. 10.7554/eLife.45399.018 Immunohistochemistry.
Figure Legend Snippet: Neonatal inflammation does not alter GFAP or IBA1 immunofluorescence in adult preBötzinger Complex or ventral cervical spinal cords. After neonatal LPS (1 mg/kg, i.p., (P4), representative confocal images (40x) from adult preBötC ( A and B ) and cervical spinal cords ( C and D ) displayed no qualitative differences in immunoreactivity for GFAP (green, astrocytes) or IBA1 (green, microglia) in males (left panels) or females (right panels). PreBötC neurons are labeled with antibodies for NK1R (red, A and B ) and motor neurons are labeled with antibodies for ChAT (red, C and D ). Neonatal inflammation did not significantly change mean fluorescent intensity of either GFAP or IBA1 in the preBötC ( E ) or cervical spinal cord ( F ), suggesting no lasting differences in astrocytes or microglia after neonatal inflammation. Scale bars: 50 µm. 10.7554/eLife.45399.018 Immunohistochemistry.

Techniques Used: Immunofluorescence, Labeling, Immunohistochemistry

29) Product Images from "Olfactory Performance as an Indicator for Protective Treatment Effects in an Animal Model of Neurodegeneration"

Article Title: Olfactory Performance as an Indicator for Protective Treatment Effects in an Animal Model of Neurodegeneration

Journal: Frontiers in Integrative Neuroscience

doi: 10.3389/fnint.2018.00035

Pronounced microgliosis in the olfactory bulb (OB) of NPC1 -/- mice, reduced by combination treatment. (A–D). Immunohistochemical reaction of Iba1(+) microglial cells in the OB. (A) Sham-treated NPC1 +/+ mice revealed no reactivity for Iba1. (B) Sham-treated NPC1 -/- demonstrated a noticeable increase of Iba1(+) cells in the granular cell layer (GCL), external plexiform layer (EPL) and glomerular layer (GL). The microgliosis was reduced after combination treatment in NPC1 -/- mice (C) . However, HPßCD treatment retained an elevated microglial activity in NPC1 -/- mice (D) . Western blot analysis of the OB of differently treated NPC1 -/- mice compared to sham-treated NPC1 +/+ mice (E) . Membrane was probed with an anti-Iba1 antibody and showed a clearly increased Iba1 signal (∼17 kDa) in sham-treated NPC1 -/- mice compared to NPC1 +/+ mice. The combination and HPßCD treatments revealed reduced microgliosis compared to sham-treated NPC1 -/- mice. ß-Actin (∼42 kDa) was used as loading control.
Figure Legend Snippet: Pronounced microgliosis in the olfactory bulb (OB) of NPC1 -/- mice, reduced by combination treatment. (A–D). Immunohistochemical reaction of Iba1(+) microglial cells in the OB. (A) Sham-treated NPC1 +/+ mice revealed no reactivity for Iba1. (B) Sham-treated NPC1 -/- demonstrated a noticeable increase of Iba1(+) cells in the granular cell layer (GCL), external plexiform layer (EPL) and glomerular layer (GL). The microgliosis was reduced after combination treatment in NPC1 -/- mice (C) . However, HPßCD treatment retained an elevated microglial activity in NPC1 -/- mice (D) . Western blot analysis of the OB of differently treated NPC1 -/- mice compared to sham-treated NPC1 +/+ mice (E) . Membrane was probed with an anti-Iba1 antibody and showed a clearly increased Iba1 signal (∼17 kDa) in sham-treated NPC1 -/- mice compared to NPC1 +/+ mice. The combination and HPßCD treatments revealed reduced microgliosis compared to sham-treated NPC1 -/- mice. ß-Actin (∼42 kDa) was used as loading control.

Techniques Used: Mouse Assay, Immunohistochemistry, Activity Assay, Western Blot

30) Product Images from "Exercise prevents obesity-induced cognitive decline and white matter damage in mice"

Article Title: Exercise prevents obesity-induced cognitive decline and white matter damage in mice

Journal: Neurobiology of aging

doi: 10.1016/j.neurobiolaging.2019.03.018

WD causes activation of phagocytosing myeloid cells. ( A, B ) A representative image of the corpus callosum from an aged WD mouse showing myelin (MBP), myeloid cells (IBA1) and phagosome-containing myeloid cells (CD68). ( C ) There was a significant increase of IBA1+ cells in the corpus callosum of aged WD compared to aged chow mice (n≥7, **p= 0.0012). ( D ) Myelin-myeloid cell interactions were also significantly increased in aged WD compared to aged chow mice (n≥5, **p= 0.0035). ( E–G ) IMARIS was used to identify myeloid cells that were actively phagocytosing myelin. The image shows activated myeloid cells interacting with myelin visualized with anti-MBP ( E ). F and G are higher resolution images from the boxed region in E and show MBP (red) contacting myeloid cells (green). Labeling in purple shows the interactions between CD68+IBA1+ cells. Arrows ( F, G ) show MBP inside the cell body of the myeloid cells.
Figure Legend Snippet: WD causes activation of phagocytosing myeloid cells. ( A, B ) A representative image of the corpus callosum from an aged WD mouse showing myelin (MBP), myeloid cells (IBA1) and phagosome-containing myeloid cells (CD68). ( C ) There was a significant increase of IBA1+ cells in the corpus callosum of aged WD compared to aged chow mice (n≥7, **p= 0.0012). ( D ) Myelin-myeloid cell interactions were also significantly increased in aged WD compared to aged chow mice (n≥5, **p= 0.0035). ( E–G ) IMARIS was used to identify myeloid cells that were actively phagocytosing myelin. The image shows activated myeloid cells interacting with myelin visualized with anti-MBP ( E ). F and G are higher resolution images from the boxed region in E and show MBP (red) contacting myeloid cells (green). Labeling in purple shows the interactions between CD68+IBA1+ cells. Arrows ( F, G ) show MBP inside the cell body of the myeloid cells.

Techniques Used: Activation Assay, Mouse Assay, Labeling

31) Product Images from "Can quantifying morphology and TMEM119 expression distinguish between microglia and infiltrating macrophages after ischemic stroke and reperfusion in male and female mice?"

Article Title: Can quantifying morphology and TMEM119 expression distinguish between microglia and infiltrating macrophages after ischemic stroke and reperfusion in male and female mice?

Journal: bioRxiv

doi: 10.1101/2020.09.23.310433

TMEM119 antibody validation in control samples. Example blots of TMEM119 (A) , IBA1 (B) , and total protein (C) in SH-SY5Y cells (lane 1), spleen (lane 2) and brain (lane 3) samples. This image illustrates that while TMEM119 is present in the SH-SY5Y cells (tested by manufacturer) and brain samples, it is not present in the spleen. IBA1 is present in all samples.
Figure Legend Snippet: TMEM119 antibody validation in control samples. Example blots of TMEM119 (A) , IBA1 (B) , and total protein (C) in SH-SY5Y cells (lane 1), spleen (lane 2) and brain (lane 3) samples. This image illustrates that while TMEM119 is present in the SH-SY5Y cells (tested by manufacturer) and brain samples, it is not present in the spleen. IBA1 is present in all samples.

Techniques Used:

Microglia morphologic response to ischemic stroke is not influenced by sex or menopause. A) Images of IBA1 positive microglia (40X objective) in the cortex of mice after sham and stroke procedure. Three cortical ipsilateral regions in proximity to the infarcted tissue were included in the analysis as depicted in (B) . Summary data show that both the number of microglia process endpoints/cell (C) and summed process length/cell (D) decreases in proximity to the brain injury but with no sex differences. Post-hoc analysis is shown in the figure (****, p
Figure Legend Snippet: Microglia morphologic response to ischemic stroke is not influenced by sex or menopause. A) Images of IBA1 positive microglia (40X objective) in the cortex of mice after sham and stroke procedure. Three cortical ipsilateral regions in proximity to the infarcted tissue were included in the analysis as depicted in (B) . Summary data show that both the number of microglia process endpoints/cell (C) and summed process length/cell (D) decreases in proximity to the brain injury but with no sex differences. Post-hoc analysis is shown in the figure (****, p

Techniques Used: Mouse Assay

TMEM119 protein expression is unchanged in proximity to the infarcted tissue. Example blots of TMEM119 (A) , IBA1 (B) , and total protein (C) for regions and sex groups. D) Summary of IBA1 (D) and TMEM119 (E) data illustrate that these proteins remain relatively unchanged among brain regions and sex groups after ischemic stroke. Sample sizes range from 3-5 among sex groups and regions and are shown in (A) .
Figure Legend Snippet: TMEM119 protein expression is unchanged in proximity to the infarcted tissue. Example blots of TMEM119 (A) , IBA1 (B) , and total protein (C) for regions and sex groups. D) Summary of IBA1 (D) and TMEM119 (E) data illustrate that these proteins remain relatively unchanged among brain regions and sex groups after ischemic stroke. Sample sizes range from 3-5 among sex groups and regions and are shown in (A) .

Techniques Used: Expressing

Percent area of TMEM119 immunofluorescence is decreased in proximity to the infarcted tissue. A) Images of IBA1 and TMEM119 immunofluorescence (20X objective) in the cortex of mice after sham and stroke procedure. Two brain regions (distal and proximal to injury) were imaged in the ipsilateral hemisphere. Below, cropped images show detail and composite show co-localization of IBA1 and TMEM119 immunofluorescence. B) Illustration of brain regions imaged. C) The percent area of IBA1 immunofluorescence per cells in frame remains unchanged among brain regions and sex. D) The percent area of TMEM119 immunofluorescence per IBA1 cells in frame is decreased in the ipsilateral proximal region versus sham in all sex groups. *, p
Figure Legend Snippet: Percent area of TMEM119 immunofluorescence is decreased in proximity to the infarcted tissue. A) Images of IBA1 and TMEM119 immunofluorescence (20X objective) in the cortex of mice after sham and stroke procedure. Two brain regions (distal and proximal to injury) were imaged in the ipsilateral hemisphere. Below, cropped images show detail and composite show co-localization of IBA1 and TMEM119 immunofluorescence. B) Illustration of brain regions imaged. C) The percent area of IBA1 immunofluorescence per cells in frame remains unchanged among brain regions and sex. D) The percent area of TMEM119 immunofluorescence per IBA1 cells in frame is decreased in the ipsilateral proximal region versus sham in all sex groups. *, p

Techniques Used: Immunofluorescence, Mouse Assay

32) Product Images from "A 3′UTR modification of the TNF-α mouse gene increases peripheral TNF-α and modulates the Alzheimer-like phenotype in 5XFAD mice"

Article Title: A 3′UTR modification of the TNF-α mouse gene increases peripheral TNF-α and modulates the Alzheimer-like phenotype in 5XFAD mice

Journal: Scientific Reports

doi: 10.1038/s41598-020-65378-2

Microglia but not astrocytes are activated in 5XFAD/TNF ΔARE/+ mice. ( A,D ) Immunofluorescent detection of Iba1-positive microglia ( A ) and GFAP-positive astrocytes ( D ) in 40μm sagittal brain sections of 4-month-old 5XFAD/TNF ΔARE/+ and 5XFAD control mice. Sections were also stained with Thioflavine-S for amyloid plaques. 5XFAD/TNF ΔARE/+ brains display increased microglial cells surrounding amyloid plaques compared to 5XFAD brains. Representative pictures of the hippocampus, the cortex and the subiculum of the hippocampus are shown for each group. Scale bars: 250μm (hippocampus and cortex); 25μm (subiculum). ( B,C,E,F ) Western blot analysis shows significantly increased Iba1 brain protein levels ( B,C ) but not GFAP ( E,F ) in 5XFAD/TNF ΔARE/+ mice compared to 5XFAD mice. ( G,H,I,J ) Similarly, Western blot analysis of TNF ΔARE/+ and control brains, show significantly increased Iba1 ( G,H ) but not GFAP ( I,J ) brain protein levels in the TNF ΔARE/+ brain protein extracts compared to the C57BL/6 control brains. Quantitation was performed with densitometric analysis using ImageJ software. Data represent mean ± SEM; n = 3 mice per group; experiments repeated 3 times. For statistical analyses, two-tailed unpaired t test was used. ** p = 0.0078 (C), ** p = 0,0016 (H) .
Figure Legend Snippet: Microglia but not astrocytes are activated in 5XFAD/TNF ΔARE/+ mice. ( A,D ) Immunofluorescent detection of Iba1-positive microglia ( A ) and GFAP-positive astrocytes ( D ) in 40μm sagittal brain sections of 4-month-old 5XFAD/TNF ΔARE/+ and 5XFAD control mice. Sections were also stained with Thioflavine-S for amyloid plaques. 5XFAD/TNF ΔARE/+ brains display increased microglial cells surrounding amyloid plaques compared to 5XFAD brains. Representative pictures of the hippocampus, the cortex and the subiculum of the hippocampus are shown for each group. Scale bars: 250μm (hippocampus and cortex); 25μm (subiculum). ( B,C,E,F ) Western blot analysis shows significantly increased Iba1 brain protein levels ( B,C ) but not GFAP ( E,F ) in 5XFAD/TNF ΔARE/+ mice compared to 5XFAD mice. ( G,H,I,J ) Similarly, Western blot analysis of TNF ΔARE/+ and control brains, show significantly increased Iba1 ( G,H ) but not GFAP ( I,J ) brain protein levels in the TNF ΔARE/+ brain protein extracts compared to the C57BL/6 control brains. Quantitation was performed with densitometric analysis using ImageJ software. Data represent mean ± SEM; n = 3 mice per group; experiments repeated 3 times. For statistical analyses, two-tailed unpaired t test was used. ** p = 0.0078 (C), ** p = 0,0016 (H) .

Techniques Used: Mouse Assay, Staining, Western Blot, Quantitation Assay, Software, Two Tailed Test

CD45 infiltrating leukocytes are increased while CD68-positive phagocytic microglia and LC3II levels show a trend towards increase in the 5XFAD/TNF ΔARE/+ brains. ( A,B,C ) Immunofluorescent detection of CD45 positive leukocytes at the subiculum of the hippocampus and the cortex (arrows) shows an increase in 5XFAD/TNF ΔARE/+ brains compared to 5XFAD controls. Representative pictures of the subiculum and the cortex ( A ) are shown for each group. Scale bars: 50 μm. Quantitation of CD45 leukocytes in the subiculum ( B ) and the cortex ( C ), reveals a significant increase in 5XFAD/TNF ΔARE/+ brains. Data are mean ± SEM; n = 3 mice per group. For statistical analyses, two-tailed unpaired t test was used. *p = 0,0269, ** p = 0,0030 ( C ). ( D ) Immunofluorescent detection of CD68/Iba1 in brain sections shows a small non-significant increase of CD68 in activated microglia surrounding amyloid plaques in 5XFAD/TNF ΔARE/+ mice compared to 5XFAD controls. Sections were also stained for amyloid plaques with Thioflavine S. Scale bars: 50 μm. ( E ) Western blot analysis of LC3 brain protein levels in 5XFAD/TNF ΔARE/+ brains reveals an increase in the LC3II form and the LC3II/LC3I ratio ( F ), suggesting an increase in autophagy compared to 5XFAD controls. Analysis was performed with densitometric analysis using ImageJ software. Data are mean ± SEM; n = 3 mice per group. For statistical analyses, two-tailed unpaired t test was used.
Figure Legend Snippet: CD45 infiltrating leukocytes are increased while CD68-positive phagocytic microglia and LC3II levels show a trend towards increase in the 5XFAD/TNF ΔARE/+ brains. ( A,B,C ) Immunofluorescent detection of CD45 positive leukocytes at the subiculum of the hippocampus and the cortex (arrows) shows an increase in 5XFAD/TNF ΔARE/+ brains compared to 5XFAD controls. Representative pictures of the subiculum and the cortex ( A ) are shown for each group. Scale bars: 50 μm. Quantitation of CD45 leukocytes in the subiculum ( B ) and the cortex ( C ), reveals a significant increase in 5XFAD/TNF ΔARE/+ brains. Data are mean ± SEM; n = 3 mice per group. For statistical analyses, two-tailed unpaired t test was used. *p = 0,0269, ** p = 0,0030 ( C ). ( D ) Immunofluorescent detection of CD68/Iba1 in brain sections shows a small non-significant increase of CD68 in activated microglia surrounding amyloid plaques in 5XFAD/TNF ΔARE/+ mice compared to 5XFAD controls. Sections were also stained for amyloid plaques with Thioflavine S. Scale bars: 50 μm. ( E ) Western blot analysis of LC3 brain protein levels in 5XFAD/TNF ΔARE/+ brains reveals an increase in the LC3II form and the LC3II/LC3I ratio ( F ), suggesting an increase in autophagy compared to 5XFAD controls. Analysis was performed with densitometric analysis using ImageJ software. Data are mean ± SEM; n = 3 mice per group. For statistical analyses, two-tailed unpaired t test was used.

Techniques Used: Quantitation Assay, Mouse Assay, Two Tailed Test, Staining, Western Blot, Software

5XFAD/TNF ΔARE/+ mice display increased CD206-positive meningeal and perivascular macrophages (arrows). ( A – C ) Double immunofluorescence for CD206/Iba1 (A) and CD206/α-SMA ( B, C ) in sagittal brain sections of 4-month-old 5XFAD and 5XFAD/TNF ΔARE/+ mice. Representative pictures of the hippocampus, cortex and cortical vessels are shown for each group. Arrowheads indicate meningeal macrophages ( A ) and perivascular CD206-positive macrophages in the cortex ( B ) and the hippocampus ( C ). Microglia is stained red ( A ) as well as blood vessels ( B, C ) Scale bars: 25 μm. ( D-G ) Western blot analysis of CD206 brain protein levels in 4-month-old 5XFAD/TNF ΔARE/+ – 5XFAD ( D ) and TNF ΔARE/+ – C57BL/6 mice ( F ) revealed an increase in 5XFAD/TNF ΔARE/+ ( E ) and TNF ΔARE/+ mice ( G ) compared with the 5XFAD and C57BL/6 control groups. Quantitation was performed with densitometric analysis using ImageJ software. Data are mean ± SEM; n = 3 mice per group; experiment repeated 3 times. For statistical analyses, two-tailed unpaired t test was used. ** p = 0.0099, * p = 0,0403.
Figure Legend Snippet: 5XFAD/TNF ΔARE/+ mice display increased CD206-positive meningeal and perivascular macrophages (arrows). ( A – C ) Double immunofluorescence for CD206/Iba1 (A) and CD206/α-SMA ( B, C ) in sagittal brain sections of 4-month-old 5XFAD and 5XFAD/TNF ΔARE/+ mice. Representative pictures of the hippocampus, cortex and cortical vessels are shown for each group. Arrowheads indicate meningeal macrophages ( A ) and perivascular CD206-positive macrophages in the cortex ( B ) and the hippocampus ( C ). Microglia is stained red ( A ) as well as blood vessels ( B, C ) Scale bars: 25 μm. ( D-G ) Western blot analysis of CD206 brain protein levels in 4-month-old 5XFAD/TNF ΔARE/+ – 5XFAD ( D ) and TNF ΔARE/+ – C57BL/6 mice ( F ) revealed an increase in 5XFAD/TNF ΔARE/+ ( E ) and TNF ΔARE/+ mice ( G ) compared with the 5XFAD and C57BL/6 control groups. Quantitation was performed with densitometric analysis using ImageJ software. Data are mean ± SEM; n = 3 mice per group; experiment repeated 3 times. For statistical analyses, two-tailed unpaired t test was used. ** p = 0.0099, * p = 0,0403.

Techniques Used: Mouse Assay, Immunofluorescence, Staining, Western Blot, Quantitation Assay, Software, Two Tailed Test

33) Product Images from "Elevated protein synthesis in microglia causes autism-like synaptic and behavioral aberrations"

Article Title: Elevated protein synthesis in microglia causes autism-like synaptic and behavioral aberrations

Journal: Nature Communications

doi: 10.1038/s41467-020-15530-3

Microglial surveillance and synapse engulfment in MG 4E mice. a Phagocytosis of FAM-Aβ (1–42) in cultured control and MG 4E microglia. Male, n = 18 for control and n = 28 for MG 4E ; female, n = 14 for control and n = 13 for MG 4E . ** p = 0.0017 and n.s. not significant ( p = 0.4919) by two-sided t -test. b , c Migration of microglia into FAM-Aβ (1–42) injection sites in 2-week-old male ( b ) and female ( c ) control and MG 4E mice. Microglia clustering index is defined as (density of Iba1 + cells in the FAM-Aβ-covered area)/(density of Iba1 + cells in a contralateral site). Male, n = 6 control mice and 7 MG 4E mice; female, n = 4 per genotype. Two-sided t -test: male mice, contralateral microglia density, ** p = 0.0013; clustering index, ** p = 0.0026, n.s. not significant. Scale bars, 100 μm. d Microglial surveillance in response to ATP treatment in male and female MG 4E microglia at P14–P18. Microglial baseline motility was recorded for 5 min, then ATP was bath-applied to brain slices and recorded for another 10 min. Data was normalized to the mean process motility of first 5 min. Male, n = 15 microglia from 3 control mice and 20 microglia from 3 MG 4E mice; female, n = 18 microglia from 3 control mice and 26 microglia from 3 MG 4E mice. Male, two-way ANOVA for genotype during ATP treatment, F (1, 330) = 9.566, p = 0.002; female, two-way ANOVA for genotype during ATP treatment, F (1, 420) = 0.045, p = 0.831. Scale bar, 10 μm. e Engulfment of Homer1 by microglia. Upper panel shows confocal images of Homer1 and Iba1 double immunohistochemistry, and lower panel shows 3-D reconstruction of a microglial cell and Homer1 immunoreactivity. Arrowheads denote Homer1 inside the microglia. Male, 18 microglia from 6 control mice and 15 microglia from 5 MG 4E mice; female, 15 microglia from 5 mice per genotype. * p = 0.0378 and n.s., not significant ( p = 0.4891) by two-sided t -test. Scale bars, 5 μm. All data are shown as mean ± s.e.m. Source data are provided as a Source Data file.
Figure Legend Snippet: Microglial surveillance and synapse engulfment in MG 4E mice. a Phagocytosis of FAM-Aβ (1–42) in cultured control and MG 4E microglia. Male, n = 18 for control and n = 28 for MG 4E ; female, n = 14 for control and n = 13 for MG 4E . ** p = 0.0017 and n.s. not significant ( p = 0.4919) by two-sided t -test. b , c Migration of microglia into FAM-Aβ (1–42) injection sites in 2-week-old male ( b ) and female ( c ) control and MG 4E mice. Microglia clustering index is defined as (density of Iba1 + cells in the FAM-Aβ-covered area)/(density of Iba1 + cells in a contralateral site). Male, n = 6 control mice and 7 MG 4E mice; female, n = 4 per genotype. Two-sided t -test: male mice, contralateral microglia density, ** p = 0.0013; clustering index, ** p = 0.0026, n.s. not significant. Scale bars, 100 μm. d Microglial surveillance in response to ATP treatment in male and female MG 4E microglia at P14–P18. Microglial baseline motility was recorded for 5 min, then ATP was bath-applied to brain slices and recorded for another 10 min. Data was normalized to the mean process motility of first 5 min. Male, n = 15 microglia from 3 control mice and 20 microglia from 3 MG 4E mice; female, n = 18 microglia from 3 control mice and 26 microglia from 3 MG 4E mice. Male, two-way ANOVA for genotype during ATP treatment, F (1, 330) = 9.566, p = 0.002; female, two-way ANOVA for genotype during ATP treatment, F (1, 420) = 0.045, p = 0.831. Scale bar, 10 μm. e Engulfment of Homer1 by microglia. Upper panel shows confocal images of Homer1 and Iba1 double immunohistochemistry, and lower panel shows 3-D reconstruction of a microglial cell and Homer1 immunoreactivity. Arrowheads denote Homer1 inside the microglia. Male, 18 microglia from 6 control mice and 15 microglia from 5 MG 4E mice; female, 15 microglia from 5 mice per genotype. * p = 0.0378 and n.s., not significant ( p = 0.4891) by two-sided t -test. Scale bars, 5 μm. All data are shown as mean ± s.e.m. Source data are provided as a Source Data file.

Techniques Used: Mouse Assay, Cell Culture, Migration, Injection, Immunohistochemistry

Iba1 immunohistochemistry reveals altered microglial density and morphology in male MG 4E mice. a – c Increased microglial density and size in the mPFC of male MG 4E mice. n = 7 control mice and 6 MG 4E mice. Ten microglia from each mouse were randomly selected for measurement of cell size (cross section area). * p = 0.0342 and *** p
Figure Legend Snippet: Iba1 immunohistochemistry reveals altered microglial density and morphology in male MG 4E mice. a – c Increased microglial density and size in the mPFC of male MG 4E mice. n = 7 control mice and 6 MG 4E mice. Ten microglia from each mouse were randomly selected for measurement of cell size (cross section area). * p = 0.0342 and *** p

Techniques Used: Immunohistochemistry, Mouse Assay

34) Product Images from "A live imaging‐friendly slice culture method using collagen membranes. A live imaging‐friendly slice culture method using collagen membranes"

Article Title: A live imaging‐friendly slice culture method using collagen membranes. A live imaging‐friendly slice culture method using collagen membranes

Journal: Neuropsychopharmacology Reports

doi: 10.1002/npr2.12128

A, Schematic diagram of the live imaging setup. Immunostained slices were imaged through the culture membrane (after fixation) using an inverted microscope. The location (starting at the bottom of the slice) where staining was first observed was defined as 0 µm. B, Representative images of immunostained slice cultures at 14 DIV (NeuN and Iba1, DNA counterstained with Hoechst); 0, 48, 60, and 72 µm
Figure Legend Snippet: A, Schematic diagram of the live imaging setup. Immunostained slices were imaged through the culture membrane (after fixation) using an inverted microscope. The location (starting at the bottom of the slice) where staining was first observed was defined as 0 µm. B, Representative images of immunostained slice cultures at 14 DIV (NeuN and Iba1, DNA counterstained with Hoechst); 0, 48, 60, and 72 µm

Techniques Used: Imaging, Inverted Microscopy, Staining

A, Representative images of the entorhinohippocampal slice cultures at 7 d in vitro (DIV) immunostained for caspase‐3. Nuclei were traced with Hoechst. B, The density of caspase‐3‐positive cells cultured on PTFE and collagen membranes. P > .05 vs PTFE membranes; Student's t test, n = 6 slices. Data represent mean ± SD. C, Representative images of the hippocampal slice cultures at 14 DIV immunostained for NeuN and Iba1 and DNA counterstained with Hoechst. D, E, The density of NeuN‐positive cells D, and Iba1‐positive cells E, at 14 DIV P > .05 vs PTFE membranes; Student's t test, n = 5‐7 slices. Data represent mean ± SD. F, Representative images of hippocampal slice cultures at 14 DIV immunostained for Iba1. G, Cumulative distribution of total microglial process length at 14 DIV P > .05 vs PTFE membranes; Kolmogorov‐Smirnov test, n = 62 processes, from 7 to 8 cells, from 3 slices
Figure Legend Snippet: A, Representative images of the entorhinohippocampal slice cultures at 7 d in vitro (DIV) immunostained for caspase‐3. Nuclei were traced with Hoechst. B, The density of caspase‐3‐positive cells cultured on PTFE and collagen membranes. P > .05 vs PTFE membranes; Student's t test, n = 6 slices. Data represent mean ± SD. C, Representative images of the hippocampal slice cultures at 14 DIV immunostained for NeuN and Iba1 and DNA counterstained with Hoechst. D, E, The density of NeuN‐positive cells D, and Iba1‐positive cells E, at 14 DIV P > .05 vs PTFE membranes; Student's t test, n = 5‐7 slices. Data represent mean ± SD. F, Representative images of hippocampal slice cultures at 14 DIV immunostained for Iba1. G, Cumulative distribution of total microglial process length at 14 DIV P > .05 vs PTFE membranes; Kolmogorov‐Smirnov test, n = 62 processes, from 7 to 8 cells, from 3 slices

Techniques Used: In Vitro, Cell Culture

35) Product Images from "Adoptive transfer of immune cells from glaucomatous mice provokes retinal ganglion cell loss in recipients"

Article Title: Adoptive transfer of immune cells from glaucomatous mice provokes retinal ganglion cell loss in recipients

Journal: Acta Neuropathologica Communications

doi: 10.1186/s40478-015-0234-y

Appearance of ocular microglia in recipients of B6 or nee lymphocytes. a Iba1 immunostained microglia in the whole mounted retina of an age-matched naïve control mouse depicting quiescent, ramified microglia. Retinal microglia in recipients of ( b ) CD19 + and ( c ) CD3 + cells donated from B6 mice appear uniformly ramified 28 days after transfer. d Horizontal sections of the optic nerve also did not reveal signs of microglial activation. Here the optic nerve of a recipient after adoptive transfer of B6 CD19 + cell fraction is shown. Numerous activated microglia are readily identified in recipients of ( e ) CD19 + or ( f ) CD3 + lymphocytes from nee donor mice (bottom row). Both early-stage microglial activation, indicated by hypertrophic somatic areas (arrows in f), and highly activated microglia (arrowhead in e) were observed. g Interactions between transferred T-lymphocytes and activated resident microglia are occasionally found. These cells are located outside the retinal vasculature (V) and are accompanied by additional, unidentified, DAPI positive endogenous cells. h Microglial activation was not observed in the optic nerve of any recipients. Scale bars in a-c, e-f is 50 μm and in d, h 20 μm
Figure Legend Snippet: Appearance of ocular microglia in recipients of B6 or nee lymphocytes. a Iba1 immunostained microglia in the whole mounted retina of an age-matched naïve control mouse depicting quiescent, ramified microglia. Retinal microglia in recipients of ( b ) CD19 + and ( c ) CD3 + cells donated from B6 mice appear uniformly ramified 28 days after transfer. d Horizontal sections of the optic nerve also did not reveal signs of microglial activation. Here the optic nerve of a recipient after adoptive transfer of B6 CD19 + cell fraction is shown. Numerous activated microglia are readily identified in recipients of ( e ) CD19 + or ( f ) CD3 + lymphocytes from nee donor mice (bottom row). Both early-stage microglial activation, indicated by hypertrophic somatic areas (arrows in f), and highly activated microglia (arrowhead in e) were observed. g Interactions between transferred T-lymphocytes and activated resident microglia are occasionally found. These cells are located outside the retinal vasculature (V) and are accompanied by additional, unidentified, DAPI positive endogenous cells. h Microglial activation was not observed in the optic nerve of any recipients. Scale bars in a-c, e-f is 50 μm and in d, h 20 μm

Techniques Used: Mouse Assay, Activation Assay, Adoptive Transfer Assay

36) Product Images from "Suppression of SNARE‐dependent exocytosis in retinal glial cells and its effect on ischemia‐induced neurodegeneration, et al. Suppression of SNARE‐dependent exocytosis in retinal glial cells and its effect on ischemia‐induced neurodegeneration"

Article Title: Suppression of SNARE‐dependent exocytosis in retinal glial cells and its effect on ischemia‐induced neurodegeneration, et al. Suppression of SNARE‐dependent exocytosis in retinal glial cells and its effect on ischemia‐induced neurodegeneration

Journal: Glia

doi: 10.1002/glia.23144

Validation of glia‐specific transgene expression in retinae of dnSNARE mice. Immunohistochemical staining of retinal slices (a) and whole‐mounts (b) demonstrating exclusive EGFP expression as reporter for dnSNARE transgene expression in astrocytes and Müller cells. (a) Micrographs of retinal slices showing EGFP‐positive glial cells, protein kinase Cα (PKCα)‐positive rod bipolar cells and neurofilament‐positive neuronal fibers of the inner retina. Cell nuclei were labeled by DAPI. (b) Micrographs of retinal whole‐mounts showing Iba1‐positive microglial cells and EGFP‐ and GFAP‐positive astrocytes. Note labelling of vessels in the GFAP channel due to unspecific binding of the secondary antibody. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; ONL = outer nuclear layer; OPL = outer plexiform layer. Scale bars, 20 µm [Color figure can be viewed at wileyonlinelibrary.com ]
Figure Legend Snippet: Validation of glia‐specific transgene expression in retinae of dnSNARE mice. Immunohistochemical staining of retinal slices (a) and whole‐mounts (b) demonstrating exclusive EGFP expression as reporter for dnSNARE transgene expression in astrocytes and Müller cells. (a) Micrographs of retinal slices showing EGFP‐positive glial cells, protein kinase Cα (PKCα)‐positive rod bipolar cells and neurofilament‐positive neuronal fibers of the inner retina. Cell nuclei were labeled by DAPI. (b) Micrographs of retinal whole‐mounts showing Iba1‐positive microglial cells and EGFP‐ and GFAP‐positive astrocytes. Note labelling of vessels in the GFAP channel due to unspecific binding of the secondary antibody. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; ONL = outer nuclear layer; OPL = outer plexiform layer. Scale bars, 20 µm [Color figure can be viewed at wileyonlinelibrary.com ]

Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining, Labeling, Binding Assay

37) Product Images from "The Role of G-Protein Receptor 84 in Experimental Neuropathic Pain"

Article Title: The Role of G-Protein Receptor 84 in Experimental Neuropathic Pain

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3558-14.2015

Nerve injury-induced microgliosis is normal in GPR84 KO mice. At 7 d ( A–C ) and 21 d ( D , E ) after PNL, there was a significant increase in Iba1- and p-p38-positive cells in the ipsilateral dorsal horn of GPR84 WT (WT PNL) and KO (KO PNL) mice compared
Figure Legend Snippet: Nerve injury-induced microgliosis is normal in GPR84 KO mice. At 7 d ( A–C ) and 21 d ( D , E ) after PNL, there was a significant increase in Iba1- and p-p38-positive cells in the ipsilateral dorsal horn of GPR84 WT (WT PNL) and KO (KO PNL) mice compared

Techniques Used: Mouse Assay

After injury, GPR84 KO mice exhibit normal peripheral macrophage recruitment but elevated anti-inflammatory marker expression in the sciatic nerve. There was a significant increase in Iba1- and CD45-positive cells in the ipsilateral (ipsi) sciatic nerve
Figure Legend Snippet: After injury, GPR84 KO mice exhibit normal peripheral macrophage recruitment but elevated anti-inflammatory marker expression in the sciatic nerve. There was a significant increase in Iba1- and CD45-positive cells in the ipsilateral (ipsi) sciatic nerve

Techniques Used: Mouse Assay, Marker, Expressing

38) Product Images from "Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling"

Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-018-1351-x

Decreased neuroinflammation in brains of KCa3.1 −/− /APP/PS1 mice. a Levels of activated microglia in CA1 areas of the mouse hippocampus were analyzed by immunostaining of the microglia marker Iba1. b Quantification of activated microglia number/0.01 mm 2 in the hippocampus ( n = 3). Data are presented as the mean ± SEM. * p
Figure Legend Snippet: Decreased neuroinflammation in brains of KCa3.1 −/− /APP/PS1 mice. a Levels of activated microglia in CA1 areas of the mouse hippocampus were analyzed by immunostaining of the microglia marker Iba1. b Quantification of activated microglia number/0.01 mm 2 in the hippocampus ( n = 3). Data are presented as the mean ± SEM. * p

Techniques Used: Mouse Assay, Immunostaining, Marker

39) Product Images from "Conditional Deletion of Prnp Rescues Behavioral and Synaptic Deficits after Disease Onset in Transgenic Alzheimer's Disease"

Article Title: Conditional Deletion of Prnp Rescues Behavioral and Synaptic Deficits after Disease Onset in Transgenic Alzheimer's Disease

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0722-17.2017

Deletion of Prnp does not have an effect on microgliosis or astrocytosis regardless of time of deletion. A , Representative immunofluorescent images of the dentate gyrus stained with anti-Iba1 antibody from the 12MD cohort at 12m+4. B , Quantification of Iba1-immunoreactive percentage area in the dentate gyrus for indicated genotypes from the 12MD cohort at 12m+4. Two slices per animal were analyzed. Data are graphed as mean ± SEM per animal, n = 8 flox- Prnp Cre −/− mice, n = 7 flox- Prnp Cre −/+ mice, n = 11 APP/PS1 flox- Prnp Cre −/− mice, n = 6 APP/PS1 flox- Prnp Cre −/+ mice. C , Quantification of Iba1-immunoreactive percentage area in the dentate gyrus for the indicated genotypes from 16MD cohort at 16m+2. Two slices per animal were analyzed. Data are graphed as mean ± SEM per animal, n = 7 flox- Prnp Cre −/− mice, n = 7 flox- Prnp Cre −/+ mice, n = 6 APP/PS1 flox- Prnp Cre −/− mice, n = 6 APP/PS1 flox- Prnp Cre −/+ mice. In both B and C , APP/PS1 flox- Prnp mice with or without Cre have significantly more Iba1-immunoreactive cells compared with WT controls (one-way ANOVA with uncorrected Fisher's LSD multiple-comparisons test; * p
Figure Legend Snippet: Deletion of Prnp does not have an effect on microgliosis or astrocytosis regardless of time of deletion. A , Representative immunofluorescent images of the dentate gyrus stained with anti-Iba1 antibody from the 12MD cohort at 12m+4. B , Quantification of Iba1-immunoreactive percentage area in the dentate gyrus for indicated genotypes from the 12MD cohort at 12m+4. Two slices per animal were analyzed. Data are graphed as mean ± SEM per animal, n = 8 flox- Prnp Cre −/− mice, n = 7 flox- Prnp Cre −/+ mice, n = 11 APP/PS1 flox- Prnp Cre −/− mice, n = 6 APP/PS1 flox- Prnp Cre −/+ mice. C , Quantification of Iba1-immunoreactive percentage area in the dentate gyrus for the indicated genotypes from 16MD cohort at 16m+2. Two slices per animal were analyzed. Data are graphed as mean ± SEM per animal, n = 7 flox- Prnp Cre −/− mice, n = 7 flox- Prnp Cre −/+ mice, n = 6 APP/PS1 flox- Prnp Cre −/− mice, n = 6 APP/PS1 flox- Prnp Cre −/+ mice. In both B and C , APP/PS1 flox- Prnp mice with or without Cre have significantly more Iba1-immunoreactive cells compared with WT controls (one-way ANOVA with uncorrected Fisher's LSD multiple-comparisons test; * p

Techniques Used: Staining, Mouse Assay

40) Product Images from "A lincRNA-p21/miR-181 family feedback loop regulates microglial activation during systemic LPS- and MPTP- induced neuroinflammation"

Article Title: A lincRNA-p21/miR-181 family feedback loop regulates microglial activation during systemic LPS- and MPTP- induced neuroinflammation

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0821-5

LincRNA-p21-mediated inflammation contributes to the death of TH + neurons in the MPTP model of PD. Ad-lincRNA-p21, Ad-lincRNA-p21-shRNA, or Ad-control were stereotaxically delivered into the right substantia nigra region of C57BL/6 mice as described in methods. One week later, mice received 4 i.p. injections of MPTP-HCl (18 mg/kg free base) at 2 h intervals or equivalent saline injections to generate acute MPTP mouse model of PD. a Representative immunofluorescence double staining for Iba1 (microglia, red) and TH (green) at the non-injection site in the substantia nigra region seven days after the last MPTP injection. Scale bar = 100 μm. b , c Stereological counting of TH-positive cells ( b ) and Iba-1-positive cells ( c ) in the entire substantia nigra are shown. n = 3 mice per group. *** P
Figure Legend Snippet: LincRNA-p21-mediated inflammation contributes to the death of TH + neurons in the MPTP model of PD. Ad-lincRNA-p21, Ad-lincRNA-p21-shRNA, or Ad-control were stereotaxically delivered into the right substantia nigra region of C57BL/6 mice as described in methods. One week later, mice received 4 i.p. injections of MPTP-HCl (18 mg/kg free base) at 2 h intervals or equivalent saline injections to generate acute MPTP mouse model of PD. a Representative immunofluorescence double staining for Iba1 (microglia, red) and TH (green) at the non-injection site in the substantia nigra region seven days after the last MPTP injection. Scale bar = 100 μm. b , c Stereological counting of TH-positive cells ( b ) and Iba-1-positive cells ( c ) in the entire substantia nigra are shown. n = 3 mice per group. *** P

Techniques Used: shRNA, Mouse Assay, Immunofluorescence, Double Staining, Injection

Related Articles

SDS Page:

Article Title: Keratan sulfate expression in microglia is diminished in the spinal cord in experimental autoimmune neuritis
Article Snippet: The soluble proteins were treated with chondroitinase ABC (1 U/ml; Seikagaku Corporation, Tokyo, Japan) in 50 mM Tris-acetate buffer (pH 8.0) at 37 °C for 1 h and 20 μ l of 2 × SDS-PAGE sample buffer (0.125 M Tris-HCl, pH 6.8; 10% 2-mercaptoethanol; 4% SDS; 10% sucrose; 0.004% bromophenol blue) was added. .. The samples were subjected to SDS-PAGE after the boiling for 10 min. After transferring the proteins to a polyvinylidene difluoride membrane (Hybond-P; GE Healthcare, Uppsala, Sweden) and blocking it with PBS-T containing 5% skimmed milk, the membrane was blotted by mouse anti-KS antibody (1 : 1000; clone 5D4, Seikagaku Corporation), rabbit anti-Iba1 antibody (1 : 1000; Wako, Tokyo, Japan), mouse anti-CD68 anticody (1 : 1000; Millipore, Bedford, MA, USA), and goat anti-CD206 antibody (1 : 1000; R & D Systems). .. Anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG conjugated with horseradish peroxidase (1 : 5000; Jackson Immunoresearch, West Grove, PA, USA) were used as secondary antibodies.

Transferring:

Article Title: Keratan sulfate expression in microglia is diminished in the spinal cord in experimental autoimmune neuritis
Article Snippet: The soluble proteins were treated with chondroitinase ABC (1 U/ml; Seikagaku Corporation, Tokyo, Japan) in 50 mM Tris-acetate buffer (pH 8.0) at 37 °C for 1 h and 20 μ l of 2 × SDS-PAGE sample buffer (0.125 M Tris-HCl, pH 6.8; 10% 2-mercaptoethanol; 4% SDS; 10% sucrose; 0.004% bromophenol blue) was added. .. The samples were subjected to SDS-PAGE after the boiling for 10 min. After transferring the proteins to a polyvinylidene difluoride membrane (Hybond-P; GE Healthcare, Uppsala, Sweden) and blocking it with PBS-T containing 5% skimmed milk, the membrane was blotted by mouse anti-KS antibody (1 : 1000; clone 5D4, Seikagaku Corporation), rabbit anti-Iba1 antibody (1 : 1000; Wako, Tokyo, Japan), mouse anti-CD68 anticody (1 : 1000; Millipore, Bedford, MA, USA), and goat anti-CD206 antibody (1 : 1000; R & D Systems). .. Anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG conjugated with horseradish peroxidase (1 : 5000; Jackson Immunoresearch, West Grove, PA, USA) were used as secondary antibodies.

Blocking Assay:

Article Title: Keratan sulfate expression in microglia is diminished in the spinal cord in experimental autoimmune neuritis
Article Snippet: The soluble proteins were treated with chondroitinase ABC (1 U/ml; Seikagaku Corporation, Tokyo, Japan) in 50 mM Tris-acetate buffer (pH 8.0) at 37 °C for 1 h and 20 μ l of 2 × SDS-PAGE sample buffer (0.125 M Tris-HCl, pH 6.8; 10% 2-mercaptoethanol; 4% SDS; 10% sucrose; 0.004% bromophenol blue) was added. .. The samples were subjected to SDS-PAGE after the boiling for 10 min. After transferring the proteins to a polyvinylidene difluoride membrane (Hybond-P; GE Healthcare, Uppsala, Sweden) and blocking it with PBS-T containing 5% skimmed milk, the membrane was blotted by mouse anti-KS antibody (1 : 1000; clone 5D4, Seikagaku Corporation), rabbit anti-Iba1 antibody (1 : 1000; Wako, Tokyo, Japan), mouse anti-CD68 anticody (1 : 1000; Millipore, Bedford, MA, USA), and goat anti-CD206 antibody (1 : 1000; R & D Systems). .. Anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG conjugated with horseradish peroxidase (1 : 5000; Jackson Immunoresearch, West Grove, PA, USA) were used as secondary antibodies.

Article Title: Platelet-Activating Factor Receptors Mediate Excitatory Postsynaptic Hippocampal Injury in Experimental Autoimmune Encephalomyelitis
Article Snippet: Free-floating sections were washed in PBS (3 × 30 min) followed by glycine (100 m m ) to reduce autofluorescence, incubated in citrate antigen unmasking solution (Vector H3300) with 0.05% Tween for 30 min at 37°C, and washed again in PBS. .. We incubated sections for 2 d at room temperature in 1.5% BSA (Sigma), 3% normal goat serum (Vector Laboratories), 0.5% Triton X-100 (Promega), and 1.8% NaCl for blocking and permeabilization, mixed with the following primary antibodies: mouse anti-PSD95 (NeuroMab 75-028, 1:500), guinea pig anti-VGLUT1 (Synaptic Signaling, 1:2500), rabbit anti-Iba1 (Wako Biochemicals 019-19741, 1:1000), rat anti-myelin basic protein (MBP, EMD Millipore, 1:500), and mouse anti-E06 (Avanti Polar Lipids, 1:500). .. We washed sections in PBS with 1.8% NaCl (3 × 30 min) and then incubated overnight at room temperature with AlexaFluor-conjugated secondary antibodies (Invitrogen, 1:500) in the same blocking/permeabilization mixture as above.

Incubation:

Article Title: Platelet-Activating Factor Receptors Mediate Excitatory Postsynaptic Hippocampal Injury in Experimental Autoimmune Encephalomyelitis
Article Snippet: Free-floating sections were washed in PBS (3 × 30 min) followed by glycine (100 m m ) to reduce autofluorescence, incubated in citrate antigen unmasking solution (Vector H3300) with 0.05% Tween for 30 min at 37°C, and washed again in PBS. .. We incubated sections for 2 d at room temperature in 1.5% BSA (Sigma), 3% normal goat serum (Vector Laboratories), 0.5% Triton X-100 (Promega), and 1.8% NaCl for blocking and permeabilization, mixed with the following primary antibodies: mouse anti-PSD95 (NeuroMab 75-028, 1:500), guinea pig anti-VGLUT1 (Synaptic Signaling, 1:2500), rabbit anti-Iba1 (Wako Biochemicals 019-19741, 1:1000), rat anti-myelin basic protein (MBP, EMD Millipore, 1:500), and mouse anti-E06 (Avanti Polar Lipids, 1:500). .. We washed sections in PBS with 1.8% NaCl (3 × 30 min) and then incubated overnight at room temperature with AlexaFluor-conjugated secondary antibodies (Invitrogen, 1:500) in the same blocking/permeabilization mixture as above.

Article Title: Interleukin-1 Mediates Neuroinflammatory Changes Associated With Diet-Induced Atherosclerosis
Article Snippet: Endogenous peroxidase activity and blocking treatment were performed as stated previously. .. Primary antibody incubation was performed overnight with goat anti-mouse VCAM-1 1:250 (R & D Systems, Abingdon, UK) or rabbit anti-Iba1 1:1000 (Wako Chemicals, Nuss, Germany). .. Sections were then incubated in appropriate biotinylated secondary antibody for 1 hour (rabbit anti-goat 1:1000 and goat anti-rabbit 1:750, Vector Laboratories, UK), followed by Vectastain ABC solution (Vector Laboratories, UK), before visualization with nickel-enhanced diaminobenzidine (50 mg/mL) (Vector Laboratories, UK).

Labeling:

Article Title: Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury
Article Snippet: .. Astrocytes were labeled with conjugated Cy3-anti-GFAP (1:500, Sigma) and microglia with rabbit anti-Iba1 (1:1,000, Wako) antibodies to identify changes in glial phenotype. .. Cre expression was analyzed with mouse anti-Cre antibody (1:100, EMD Millipore).

other:

Article Title: Loss of astrocyte connexins 43 and 30 does not significantly alter susceptibility or severity of acute experimental autoimmune encephalomyelitis in mice
Article Snippet: Primary antibodies were as follows: CD45 (Serotec rat monoclonal 1:500), mouse anti-GFAP (1:100, Dako, for DAB), rabbit anti-Iba1 (1:500, Wako), SMI32 (1:2000, Covance, for nonphosphorylated neurofilament H), MBP (1:2000, Covance, clone SMI99), rabbit anti-Cx43 (1:1000, Sigma); rabbit anti-Cx43 (1:100, Cell Signaling); mouse anti-Cx43 (1:250, Chemicon); mouse anti-GFAP (1:2000, Cell Signaling cat. #3670), rat anti-GFAP (Invitrogen cat. #130300, 1:100).

Staining:

Article Title: Glycolic acid protects neurons against ischemia in vitro and in two animal models of stroke
Article Snippet: .. For the GFAP and Iba1 staining of brain sections the same process was followed but the primary antibodies were goat anti-GFAP (ab53554, abcam USA) in 1:750 dilution and rabbit anti-Iba1 (019-19741, FUJIFILM Wako Pure Chemical Corporation USA) in 1:750 dilution. .. The secondary antibodies used were donkey anti-goat Alexa 555 (A-21432 Invitrogen, USA) for the GFAP labeling and donkey anti-rabbit Alexa 488 (A-21206, Invitrogen, USA) for the Iba1 labeling.

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    FUJIFILM rabbit anti iba1
    GA mitigates the increase in the amount of astrocytes and microglia in large infarcts after MCAo. A) Fluorescence microscopy images showing the staining for microglia <t>(IBA1,</t> green) and astrocytes (GFAP, red) in a sham brain section (i), as well as a vehicle (ii) and GA brain section with large infarcts. iv) Same brain section as in (iii) indicating the outlines of the different areas in which Mean Fluorescence Intensity (MFI) was measured. B) i) MFI of IBA1 staining in the ischemic area showing no significant differences between groups (1-way ANOVA, p=0,1482) ii) When large infarcts ( > 18% of hemispheric volume) are analyzed separately, the MFI of IBA1 inside the infarct area is significantly increased in the vehicle group compared to sham (mean diff. =-11,31, p
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    99
    FUJIFILM rabbit anti ionized calcium binding adaptor molecule 1 iba 1 antibody
    Intravenous MSC reduced proliferation of microglia after global HI. ( A ) Immunohistochemical <t>IBA-1</t> staining in the SCWM of the four experimental groups with squares in the first panel indicating the regions where immunoreactivity was assessed. Global HI induced a profound increase of IBA-1 immunoreactivity, which was significantly reduced by intravenous MSC treatment. ( B ) Immunohistochemical IBA-1 staining in the hippocampus of the four experimental groups. Profound proliferation of microglia was observed in the hippocampus following global HI. MSC partially reduced the inflammatory response of microglia in the hippocampus after global HI. ( C – D ) Graphical presentation of area fraction of IBA-1 immunoreactivity in SCWM and hippocampus; ( C ) geometric means ± 95% CI and ( D ) means ± 95% CI and levels of significance are depicted, which were calculated by the random intercept model with all repeated measures (i.e. brain sections) per animal (sham-SAL n=6, sham-MSC n=3, HI-SAL n=6, HI-MSC n=6). Dots show the averaged results of the repeated measures (i.e. brain sections) per animal. * P ≤0.05, ‡ P ≤0.01, # P ≤0.001. IBA-1 = ionized calcium binding adaptor molecule 1, HI = hypoxia-ischemia, SAL = saline, MSC = mesenchymal stem cells, IR = immunoreactivity. ( A – B ) Scale bars represent 1 mm.
    Rabbit Anti Ionized Calcium Binding Adaptor Molecule 1 Iba 1 Antibody, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GA mitigates the increase in the amount of astrocytes and microglia in large infarcts after MCAo. A) Fluorescence microscopy images showing the staining for microglia (IBA1, green) and astrocytes (GFAP, red) in a sham brain section (i), as well as a vehicle (ii) and GA brain section with large infarcts. iv) Same brain section as in (iii) indicating the outlines of the different areas in which Mean Fluorescence Intensity (MFI) was measured. B) i) MFI of IBA1 staining in the ischemic area showing no significant differences between groups (1-way ANOVA, p=0,1482) ii) When large infarcts ( > 18% of hemispheric volume) are analyzed separately, the MFI of IBA1 inside the infarct area is significantly increased in the vehicle group compared to sham (mean diff. =-11,31, p

    Journal: bioRxiv

    Article Title: Glycolic acid protects neurons against ischemia in vitro and in two animal models of stroke

    doi: 10.1101/2020.11.24.396051

    Figure Lengend Snippet: GA mitigates the increase in the amount of astrocytes and microglia in large infarcts after MCAo. A) Fluorescence microscopy images showing the staining for microglia (IBA1, green) and astrocytes (GFAP, red) in a sham brain section (i), as well as a vehicle (ii) and GA brain section with large infarcts. iv) Same brain section as in (iii) indicating the outlines of the different areas in which Mean Fluorescence Intensity (MFI) was measured. B) i) MFI of IBA1 staining in the ischemic area showing no significant differences between groups (1-way ANOVA, p=0,1482) ii) When large infarcts ( > 18% of hemispheric volume) are analyzed separately, the MFI of IBA1 inside the infarct area is significantly increased in the vehicle group compared to sham (mean diff. =-11,31, p

    Article Snippet: For the GFAP and Iba1 staining of brain sections the same process was followed but the primary antibodies were goat anti-GFAP (ab53554, abcam USA) in 1:750 dilution and rabbit anti-Iba1 (019-19741, FUJIFILM Wako Pure Chemical Corporation USA) in 1:750 dilution.

    Techniques: Fluorescence, Microscopy, Staining

    Levels of microglial activation differed between ApoE −/− mice fed a Paigen or Western diet. Levels of microglial activation were assessed through the immunostaining of Iba1. Microglial activation was assessed throughout the striatum, and the location of images shown is indicated in the schematic diagram. Levels of microglial activation differed for each diet, but IL-1β neutralization had no effect. Error bars indicate standard error (n=7), scale bars 200 μm and 10 μm.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Interleukin-1 Mediates Neuroinflammatory Changes Associated With Diet-Induced Atherosclerosis

    doi: 10.1161/JAHA.112.002006

    Figure Lengend Snippet: Levels of microglial activation differed between ApoE −/− mice fed a Paigen or Western diet. Levels of microglial activation were assessed through the immunostaining of Iba1. Microglial activation was assessed throughout the striatum, and the location of images shown is indicated in the schematic diagram. Levels of microglial activation differed for each diet, but IL-1β neutralization had no effect. Error bars indicate standard error (n=7), scale bars 200 μm and 10 μm.

    Article Snippet: Primary antibody incubation was performed overnight with goat anti-mouse VCAM-1 1:250 (R & D Systems, Abingdon, UK) or rabbit anti-Iba1 1:1000 (Wako Chemicals, Nuss, Germany).

    Techniques: Activation Assay, Mouse Assay, Western Blot, Immunostaining, Neutralization

    Vascular activation, microglial activation, and leukocyte accumulation are reduced in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet. Activated microglia as identified by increased Iba1 immunopositivity, thickened processes, and irregular cell bodies were seen in ApoE −/− mice fed a Paigen diet. Microglial activation was significantly reduced, to control levels, in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (A). Vascular activation was assessed through the immunostaining of the adhesion molecule VCAM. Atherosclerotic ApoE −/− mice show increased vascular activation, which is significantly reduced in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (B). Leukocyte accumulation as shown by CD45 immunostaining was increased in ApoE −/− mice fed a Paigen diet. Leukocyte accumulation was significantly reduced, to control levels, in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (C). D, Quantification of Iba1-positive microglia. E, Quantification of VCAM-positive blood vessels. F, Quantification of CD45-positive leukocytes. The “vehicle” data in F were not normally distributed; therefore, in addition to one-way ANOVA ( P =0.017) followed by Bonferroni multiple-comparison post-test, nonparametric Kruskal-Wallis test ( P =0.0065) followed by Dunn multiple-comparison test also were performed. Post-hoc comparisons gave identical results. A through F: n=6–10. G, Voluntary wheel running actogram for ApoE −/− and ApoE −/− /IL-1R1 −/− mice (n=2–3). Error bars represent standard error, * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Interleukin-1 Mediates Neuroinflammatory Changes Associated With Diet-Induced Atherosclerosis

    doi: 10.1161/JAHA.112.002006

    Figure Lengend Snippet: Vascular activation, microglial activation, and leukocyte accumulation are reduced in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet. Activated microglia as identified by increased Iba1 immunopositivity, thickened processes, and irregular cell bodies were seen in ApoE −/− mice fed a Paigen diet. Microglial activation was significantly reduced, to control levels, in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (A). Vascular activation was assessed through the immunostaining of the adhesion molecule VCAM. Atherosclerotic ApoE −/− mice show increased vascular activation, which is significantly reduced in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (B). Leukocyte accumulation as shown by CD45 immunostaining was increased in ApoE −/− mice fed a Paigen diet. Leukocyte accumulation was significantly reduced, to control levels, in ApoE −/− /IL-1R1 −/− mice fed a Paigen diet (C). D, Quantification of Iba1-positive microglia. E, Quantification of VCAM-positive blood vessels. F, Quantification of CD45-positive leukocytes. The “vehicle” data in F were not normally distributed; therefore, in addition to one-way ANOVA ( P =0.017) followed by Bonferroni multiple-comparison post-test, nonparametric Kruskal-Wallis test ( P =0.0065) followed by Dunn multiple-comparison test also were performed. Post-hoc comparisons gave identical results. A through F: n=6–10. G, Voluntary wheel running actogram for ApoE −/− and ApoE −/− /IL-1R1 −/− mice (n=2–3). Error bars represent standard error, * P

    Article Snippet: Primary antibody incubation was performed overnight with goat anti-mouse VCAM-1 1:250 (R & D Systems, Abingdon, UK) or rabbit anti-Iba1 1:1000 (Wako Chemicals, Nuss, Germany).

    Techniques: Activation Assay, Mouse Assay, Immunostaining

    Ephrin-B1 immunoreactivity was significantly upregulated in reactive astrocytes in the hippocampus following moderate CCI. (a–d) Fluorescent images show GFAP-positive astrocytes (GFAP, red in a and d, gray in c), Iba1-positive microglia (Iba1, blue in a), and ephrin-B1 immunoreactivity (ephrin-B1, green in a, gray in b) in the SR area of the CA1 hippocampus in control, 1, 3, and 7 dpi. (d) High magnification images show examples of ephrin-B1-positive astrocytes. Note that ephrin-B1-positive immunoreactivity is found in the dendrites of CA1 neurons and astrocytes in SR area of CA1 hippocampus. (e–g) Graphs show GFAP immunoreactivity per GFAP-positive astrocyte (e) or ephrin-B1 immunoreactivity per ephrin-B1-positive astrocyte (f) in control ( n = 682 cells, 9 images, 3 mice), 1 dpi ( n = 385 cells, 5 images, 3 mice), 3 dpi ( n = 732 cells, 11 images, 4 mice), and 7 dpi ( n = 1217 cells, 9 images, 3 mice) or post-sham ( n = 300–500 cells, 4–6 images, 3 mice). Note that ephrin-B1 immunoreactivity was not detected in all GFAP-positive cells. Error bars indicate SEM . Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc analysis (e,f, n = 3–4 mice, * p

    Journal: ASN NEURO

    Article Title: Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury

    doi: 10.1177/1759091416630220

    Figure Lengend Snippet: Ephrin-B1 immunoreactivity was significantly upregulated in reactive astrocytes in the hippocampus following moderate CCI. (a–d) Fluorescent images show GFAP-positive astrocytes (GFAP, red in a and d, gray in c), Iba1-positive microglia (Iba1, blue in a), and ephrin-B1 immunoreactivity (ephrin-B1, green in a, gray in b) in the SR area of the CA1 hippocampus in control, 1, 3, and 7 dpi. (d) High magnification images show examples of ephrin-B1-positive astrocytes. Note that ephrin-B1-positive immunoreactivity is found in the dendrites of CA1 neurons and astrocytes in SR area of CA1 hippocampus. (e–g) Graphs show GFAP immunoreactivity per GFAP-positive astrocyte (e) or ephrin-B1 immunoreactivity per ephrin-B1-positive astrocyte (f) in control ( n = 682 cells, 9 images, 3 mice), 1 dpi ( n = 385 cells, 5 images, 3 mice), 3 dpi ( n = 732 cells, 11 images, 4 mice), and 7 dpi ( n = 1217 cells, 9 images, 3 mice) or post-sham ( n = 300–500 cells, 4–6 images, 3 mice). Note that ephrin-B1 immunoreactivity was not detected in all GFAP-positive cells. Error bars indicate SEM . Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc analysis (e,f, n = 3–4 mice, * p

    Article Snippet: Astrocytes were labeled with conjugated Cy3-anti-GFAP (1:500, Sigma) and microglia with rabbit anti-Iba1 (1:1,000, Wako) antibodies to identify changes in glial phenotype.

    Techniques: Mouse Assay

    Intravenous MSC reduced proliferation of microglia after global HI. ( A ) Immunohistochemical IBA-1 staining in the SCWM of the four experimental groups with squares in the first panel indicating the regions where immunoreactivity was assessed. Global HI induced a profound increase of IBA-1 immunoreactivity, which was significantly reduced by intravenous MSC treatment. ( B ) Immunohistochemical IBA-1 staining in the hippocampus of the four experimental groups. Profound proliferation of microglia was observed in the hippocampus following global HI. MSC partially reduced the inflammatory response of microglia in the hippocampus after global HI. ( C – D ) Graphical presentation of area fraction of IBA-1 immunoreactivity in SCWM and hippocampus; ( C ) geometric means ± 95% CI and ( D ) means ± 95% CI and levels of significance are depicted, which were calculated by the random intercept model with all repeated measures (i.e. brain sections) per animal (sham-SAL n=6, sham-MSC n=3, HI-SAL n=6, HI-MSC n=6). Dots show the averaged results of the repeated measures (i.e. brain sections) per animal. * P ≤0.05, ‡ P ≤0.01, # P ≤0.001. IBA-1 = ionized calcium binding adaptor molecule 1, HI = hypoxia-ischemia, SAL = saline, MSC = mesenchymal stem cells, IR = immunoreactivity. ( A – B ) Scale bars represent 1 mm.

    Journal: PLoS ONE

    Article Title: Mesenchymal Stem Cells Induce T-Cell Tolerance and Protect the Preterm Brain after Global Hypoxia-Ischemia

    doi: 10.1371/journal.pone.0073031

    Figure Lengend Snippet: Intravenous MSC reduced proliferation of microglia after global HI. ( A ) Immunohistochemical IBA-1 staining in the SCWM of the four experimental groups with squares in the first panel indicating the regions where immunoreactivity was assessed. Global HI induced a profound increase of IBA-1 immunoreactivity, which was significantly reduced by intravenous MSC treatment. ( B ) Immunohistochemical IBA-1 staining in the hippocampus of the four experimental groups. Profound proliferation of microglia was observed in the hippocampus following global HI. MSC partially reduced the inflammatory response of microglia in the hippocampus after global HI. ( C – D ) Graphical presentation of area fraction of IBA-1 immunoreactivity in SCWM and hippocampus; ( C ) geometric means ± 95% CI and ( D ) means ± 95% CI and levels of significance are depicted, which were calculated by the random intercept model with all repeated measures (i.e. brain sections) per animal (sham-SAL n=6, sham-MSC n=3, HI-SAL n=6, HI-MSC n=6). Dots show the averaged results of the repeated measures (i.e. brain sections) per animal. * P ≤0.05, ‡ P ≤0.01, # P ≤0.001. IBA-1 = ionized calcium binding adaptor molecule 1, HI = hypoxia-ischemia, SAL = saline, MSC = mesenchymal stem cells, IR = immunoreactivity. ( A – B ) Scale bars represent 1 mm.

    Article Snippet: Free floating sections at the level of mid-thalamus and posterior hippocampus were stained with a rabbit anti-ionized calcium binding adaptor molecule 1 (IBA-1) antibody (Wako Pure Chemical Industries, Osaka, Japan), which is a highly specific marker for microglia, localizing resting and activated microglia [ ].

    Techniques: Immunohistochemistry, Staining, Hi-C, Binding Assay