Journal: Neurobiology of disease

Article Title: Gene co-expression networks identify Trem2 and Tyrobp as major hubs in human APOE expressing mice following traumatic brain injury

doi: 10.1016/j.nbd.2017.05.006

Figure Lengend Snippet: ME Brown correlates to TBI and is associated with Immune Response. (A) Expression barplot shows the gene expression and eigengene expression within each sample. Within the heatmap, the rows denote genes and the columns correspond to samples, with the corresponding module eigengene value for each sample shown in the bar plot below. Red denotes over-expression and green under-expression of the gene within the sample. (B) Network of genes connected to hub genes Trem2, Tyrobp, Clec7a, Cd68, Cx3cr1 representing immune response. Size of each gene was determined by modular membership value, and the weight determined edge width. (C–D) Average expression according to mRNA-seq results of genes modulated by TBI in “Immune response” category for (C) APOE3 and (D) APOE4 mice. The average expression was calculated as fold of Sham reads per million for each gene. Statistics is by edgeR, p<0.05. (E–F) Validation of mRNA-seq results for Trem2, Tyrobp, Cx3cr1, Tgfb1, Tgfbr1 for (E) APOE3 and (F) APOE4 mice by qPCR. Statistics was determined by t-test.

Article Snippet: Sections were incubated in TREM2 primary antibody (1:100, AF179, R&D Systems) overnight at 4 °C, followed by washing in PBS and labeling with biotinylated secondary antibody (1:1000, donkey α sheep; {"type":"entrez-nucleotide","attrs":{"text":"A16045","term_id":"489846","term_text":"A16045"}} A16045 ) and developed with Vectastain ABC Elite kit (Vector Laboratories) and DAB substrate.

Techniques: Expressing, Gene Expression, Over Expression, Biomarker Discovery

Journal: Neurobiology of disease

Article Title: Gene co-expression networks identify Trem2 and Tyrobp as major hubs in human APOE expressing mice following traumatic brain injury

doi: 10.1016/j.nbd.2017.05.006

Figure Lengend Snippet: TREM2 protein level and Astrocytosis are increased by TBI. Immunohistochemistry with anti-TREM2 antibody or anti-GFAP was performed in both sham and TBI mice (n=3/group). Percent intensity of Trem2 staining or GFAP staining was determined in the ipsilateral hemispheres. (A) Sham animals had low to no levels in Trem2. (B) APOE3 TBI and (C) APOE4 TBI animals had significantly higher levels of Trem2 when compared to their sham counterparts (p<0.01 for both APOE3 and APOE4). (D) Analysis of object area fraction demonstrates a significant main effect of injury (p<0.001), but not APOE isoform in Trem2 levels. Statistics is by Two-way ANOVA with post-hoc Tukey’s multiple comparisons test. (E) Sham animals demonstrated low GFAP staining levels. (F) APOE3 and (G) APOE4 TBI animals show increased GFAP staining compared to their sham counterparts, particularly near the injury site (APOE3: p<0.01; APOE4: p<0.001). Insets taken from the injury visualize the increased staining at higher magnification (20X). (H) Analysis of object area fraction demonstrates a significant main effect of injury (p<0.001), but not APOE isoform in GFAP levels. There was no significant difference between APOE3 and APOE4 animals, regardless of injury. Statistics is by Two-way ANOVA with post-hoc Tukey’s multiple comparisons test.

Article Snippet: Sections were incubated in TREM2 primary antibody (1:100, AF179, R&D Systems) overnight at 4 °C, followed by washing in PBS and labeling with biotinylated secondary antibody (1:1000, donkey α sheep; {"type":"entrez-nucleotide","attrs":{"text":"A16045","term_id":"489846","term_text":"A16045"}} A16045 ) and developed with Vectastain ABC Elite kit (Vector Laboratories) and DAB substrate.

Techniques: Immunohistochemistry, Staining

Journal: Nature Neuroscience

Article Title: Derivation and transcriptional reprogramming of border-forming wound repair astrocytes after spinal cord injury or stroke in mice

doi: 10.1038/s41593-024-01684-6

Figure Lengend Snippet: a . Top GO Biological Processes associated with 158 cARGs upregulated at all times after SCI. b . Immunohistochemistry of S100a6 and Lgals3 protein in lesion border astrocytes (LBA) after stroke (S100a6) or SCI (Lgals3). Note that while S100a6 is present in the processes of most LBA, Lgals3 is present in some but not others. c,d . Heatmaps of mean log 2 FC after SCI of 20 cARGs that are either most enriched ( c ) or most de-enriched ( d ) in astrocytes compared with other cells; graphs compare log 2 FC with mean log 2 FE in selected examples. e . Comparison of expression (FPKM) of Tyrobp and Trem2 by astrocytes and other cells at different times after SCI. Note that between 5, 14 and 28 days after SCI, expression levels of Tyrobp and Trem2 increase markedly in other cells while simultaneously decreasing markedly in astrocytes. Such a divergence in DEG levels would not be observed if Tyrobp and Trem2 levels in astrocytes were an artifact caused by the non-specific contamination of RiboTag IP samples by highly expressed transcripts derived from other cells. f . Expression (FPKM) by astrocytes and other cells of chondroitin sulphate proteoglycans (CSPGs) after SCI. Note that the prototypical CSPG, Acan , is not detectably expressed by astrocytes at any time after SCI, and Vcan and Csp4 were far more highly expressed by non-astrocytes. Only Bcan and Ncan are more highly expressed by astrocytes, but Bcan expression declines after SCI to levels below healthy, and although Ncan expression increases at 5 days after SCI, by 28 days it also declined to levels lower than in healthy astrocytes. Line plots are mean values +/- SEM where n = 4 mice for uninjured and all post SCI timepoints except 2d which was n = 5. P-Values in (a) calculated by two-sided Fisher’s exact test.

Article Snippet: Primary antibodies used included goat anti-A2m (1:300, AF1938; R&D Systems), rabbit anti-Aldh1l1 (1:1,000, Ab87117; Abcam), sheep anti-BrdU (1:800, NB-500-235; Novus), rat anti-C3 (1:400, NB200-540; Novus), goat anti-CD13 (1:600, AF2335; R&D Systems), rat anti-Cd44 (1:400, 14-0441-82; Invitrogen), rat anti-CD68 (1:1,000, MCA1957; Biorad), rabbit anti-Cd74 (1:200, A13958; Abclonal), rabbit anti-Cdsn (1:800,13184-1-AP; Proteintech), goat anti-Cxcl10 (1:200, AF-466; Novus), rabbit anti-Dnali1 (1:500, 17601-1-AP; Proteintech), rabbit anti-Fxyd1 (1:800, A15082; Abclonal), rabbit anti-GFAP (1:2,000, GA524, Z033401-2; Dako/Agilent), rat anti-GFAP (1:1,000, 13-0300; ThermoFisher), rabbit anti-hemagglutinin (1:1,000, H6908; Sigma-Aldrich), goat anti-Gpc5 (1:200, AF2607; R&D Systems), rabbit anti-Gpx1 (1:200, 29329-1-AP; Proteintech), goat anti-hemagglutinin (1:800, NB600-362; Novus Biologicals), rabbit anti-H2-Ab1 (1:200, A18658; Abclonal), rabbit anti-Hpse (1:200, 24529-1-AP; Proteintech), guinea pig anti-Iba1 (1:1,000, 234004; Synaptic Systems), rabbit anti-Iba-1 (1:800, 019-19741; Wako), rabbit anti-Id3 (1:500, 9837; Cell Signaling), rabbit anti-Kcnj10 (Kir4.1) (1:400, APC-035; Alomone Labs), rat anti-Lgals3 (1:200, 14-5301-82; ThermoFisher), rabbit anti-Lxn (1:500, 13056-1-AP; Proteintech), rabbit anti-Mfge8 (1:200, A12322; Abclonal), rabbit anti-Mmp12 (1:200, 22989-1-AP; Proteintech), goat anti-Myoc (1:400, AF2537; Novus), guinea pig anti-NeuN (1:1,000, 266004; Synaptic Systems), rabbit anti-NeuN (1:1,000, ab177487; Abcam), guinea pig anti-Olig2 (1:800, ABE1024; Millipore), rabbit anti-Olig2 (1:200, AB9610; Millipore), rabbit anti-Padi2 (1:300,12110-1-AP; Proteintech), rabbit anti-Prdx6 (1:500, 13585-1-AP; Proteintech), sheep anti-S100a6 (1:300, AF4584; R&D Systems), rabbit anti-S100a6 (1:200, A3461; Abclonal), goat anti-Serpina3n (1:200, AF4709; R&D Systems), goat anti-Sox9 (1:800, AF3075; R&D Systems), rabbit anti-Sox9 (1:800, 702016; ThermoFisher), goat anti-Sox10 (1:500, AF2864; R&D Systems), guinea pig anti-tdT (RFP) (1:1,500, 390-004; Synaptic Systems), rabbit anti-RFP (1:1,500, 600-401-379; Rockland), rabbit anti-Timp1 (1:800, 16644-1-AP; Proteintech), sheep anti-Trem2 (1:400, AF1729; Novus), rabbit anti-Tyrobp (1:400,12492S; Cell Signaling) and rat anti-Vim (1:200, MAB2105; Novus).

Techniques: Immunohistochemistry, Comparison, Expressing, Derivative Assay

Journal: Nature Neuroscience

Article Title: Derivation and transcriptional reprogramming of border-forming wound repair astrocytes after spinal cord injury or stroke in mice

doi: 10.1038/s41593-024-01684-6

Figure Lengend Snippet: a , Violin plots of snRNA-seq detected genes enriched in reactive astrocyte clusters compared with uninjured. b , IHC of proteins Tyrobp and Trem2 in LBAs after SCI. c , d , Selected astrocyte DEGs exhibiting different patterns of expression changes in the form of acute rise followed by decline ( c ) or delayed but persistent increase ( d ) after SCI as detected by Astro-RiboTag RNA-seq. e , f , g , IHC of proteins C3 ( e ), S100a6 ( f ) and Prdx6 ( g ) in LBAs after SCI. Boxes show locations of expanded regions. h , Scatterplot comparing mean log 2 FC and mean FPKM of 1,927 pARGs upregulated at least twofold from 28 to 70 days after SCI as detected by Astro-RiboTag RNA-seq. i , Scatterplot comparing mean log 2 FC and mean log 2 FE of 1,927 pARGs upregulated from 28 to 70 days after SCI. j , GO-BPs associated with 1,927 pARGs upregulated at least twofold from 28 to 70 days after SCI. k , Summary schematic showing local astrocyte responses to CNS tissue damage by dedifferentiation, proliferation and transcriptional reprogramming into border-forming wound repair astrocytes. Line plots are mean values; error bars, s.e.m.; n = 4 mice for uninjured and all post-SCI timepoints except day 2 ( n = 5). P values in ( j ) calculated by two-sided Fisher’s exact test.

Article Snippet: Primary antibodies used included goat anti-A2m (1:300, AF1938; R&D Systems), rabbit anti-Aldh1l1 (1:1,000, Ab87117; Abcam), sheep anti-BrdU (1:800, NB-500-235; Novus), rat anti-C3 (1:400, NB200-540; Novus), goat anti-CD13 (1:600, AF2335; R&D Systems), rat anti-Cd44 (1:400, 14-0441-82; Invitrogen), rat anti-CD68 (1:1,000, MCA1957; Biorad), rabbit anti-Cd74 (1:200, A13958; Abclonal), rabbit anti-Cdsn (1:800,13184-1-AP; Proteintech), goat anti-Cxcl10 (1:200, AF-466; Novus), rabbit anti-Dnali1 (1:500, 17601-1-AP; Proteintech), rabbit anti-Fxyd1 (1:800, A15082; Abclonal), rabbit anti-GFAP (1:2,000, GA524, Z033401-2; Dako/Agilent), rat anti-GFAP (1:1,000, 13-0300; ThermoFisher), rabbit anti-hemagglutinin (1:1,000, H6908; Sigma-Aldrich), goat anti-Gpc5 (1:200, AF2607; R&D Systems), rabbit anti-Gpx1 (1:200, 29329-1-AP; Proteintech), goat anti-hemagglutinin (1:800, NB600-362; Novus Biologicals), rabbit anti-H2-Ab1 (1:200, A18658; Abclonal), rabbit anti-Hpse (1:200, 24529-1-AP; Proteintech), guinea pig anti-Iba1 (1:1,000, 234004; Synaptic Systems), rabbit anti-Iba-1 (1:800, 019-19741; Wako), rabbit anti-Id3 (1:500, 9837; Cell Signaling), rabbit anti-Kcnj10 (Kir4.1) (1:400, APC-035; Alomone Labs), rat anti-Lgals3 (1:200, 14-5301-82; ThermoFisher), rabbit anti-Lxn (1:500, 13056-1-AP; Proteintech), rabbit anti-Mfge8 (1:200, A12322; Abclonal), rabbit anti-Mmp12 (1:200, 22989-1-AP; Proteintech), goat anti-Myoc (1:400, AF2537; Novus), guinea pig anti-NeuN (1:1,000, 266004; Synaptic Systems), rabbit anti-NeuN (1:1,000, ab177487; Abcam), guinea pig anti-Olig2 (1:800, ABE1024; Millipore), rabbit anti-Olig2 (1:200, AB9610; Millipore), rabbit anti-Padi2 (1:300,12110-1-AP; Proteintech), rabbit anti-Prdx6 (1:500, 13585-1-AP; Proteintech), sheep anti-S100a6 (1:300, AF4584; R&D Systems), rabbit anti-S100a6 (1:200, A3461; Abclonal), goat anti-Serpina3n (1:200, AF4709; R&D Systems), goat anti-Sox9 (1:800, AF3075; R&D Systems), rabbit anti-Sox9 (1:800, 702016; ThermoFisher), goat anti-Sox10 (1:500, AF2864; R&D Systems), guinea pig anti-tdT (RFP) (1:1,500, 390-004; Synaptic Systems), rabbit anti-RFP (1:1,500, 600-401-379; Rockland), rabbit anti-Timp1 (1:800, 16644-1-AP; Proteintech), sheep anti-Trem2 (1:400, AF1729; Novus), rabbit anti-Tyrobp (1:400,12492S; Cell Signaling) and rat anti-Vim (1:200, MAB2105; Novus).

Techniques: Expressing, RNA Sequencing Assay

Journal: Scientific reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies.

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Fig. 4. DOX withdrawal promotes microglial differentiation and increases expression of microglial genes TREM2, P2RY12, CD45, and CD11b. (a) Timeline of experiments for doxycycline induction (DOX) and control (CTRL) treatment groups. (b) Representative images of microglial cells at day 4: the left panel shows doxycycline-induced immortalization, while the right panel shows control cells. The top panel displays IBA1 expression, the middle panel highlights increased TREM2, and the bottom panel shows elevated P2RY12 expression following doxycycline withdrawal (scale bar = 50 μm). (c) Histogram comparing CD45 and CD11b expression in the immortalized BV-2 cell line (dark green) versus transduced microglia treated with doxycycline (DOX, red), no doxycycline (CTRL, blue), or with 20 ng/ml M-CSF (orange) or TGFb (bright green) over three days. (d) Percentage of CD45 + CD11b + microglial cells based on treatment. (e) Quantification of mean fluorescence intensity (MFI) for CD45 and CD11b. N = 3 replicates from 2 independent batches. (f) RNA sequencing results for microglial markers Aif1, Trem2, and P2ry12, presented in counts per million (CPM) on a log2 scale. N = 6 replicates from 3 independent batches. Statistical analyses were performed using One-way ANOVA followed by Tukey’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent mean ± SEM.

Article Snippet: The following primary antibodies were used: TREM2 (Bio-Rad AF1729) and rabbit-IBA1 (WAKO 019–19741).

Techniques: Expressing, Control, Fluorescence, RNA Sequencing, Comparison