sheep  (R&D Systems)

Journal: bioRxiv

Article Title: Apolipoprotein E ε4 exacerbates microglia-mediated complement-dependent synapse loss caused by neuronal Tpk deficiency

doi: 10.1101/2025.01.16.633468

Figure Lengend Snippet: a. Flow chart of injection of SB290157. The injection of SB290157 started from the 4 weeks after tamoxifen induction, the frequency of injections is 3 times per week. b. Scores of nest building. The results showed that the injection of SB290157 did not alleviate the abilities of nest building of the AE-cKO mice. N = 5 per group. c. Rotarod test. The results showed that the injection of SB290157 did not alleviate the run time on the rotarod of the AE-cKO mice. N = 5 per group. d. Y-maze test. The results showed that the injection of SB290157 did not alleviate the behavior abnormality of the AE-cKO mice. N = 5 per group. e-g. Representative images of Iba1 (red) immunostaining ( e ) and quantification ( f,g ) in the cortices of mice. The results showed that the injection of SB290157 significantly decreased the activation of microglia in the cortices of the AE-cKO mice, but did not affect the count of Iba. h, i. Representative images of Iba1 (red) and CD68 (green) immunostaining ( h ) and quantification ( i ) in the cortices of mice. The results showed that the injection of SB290157 significantly decreased the elevation of co-labeled cells stained by Iba1 and CD68 antibodies in the AE-cKO mice. j, k. Representative images of Iba1 (red) and TSPO (green) immunostaining ( j ) and quantification ( k ) in the cortices of mice. The results showed that the injection of SB290157 significantly downregulated the enhancement of co-labeled cells stained by Iba1 and TSPO antibodies in the AE-cKO mice. l, m. Representative images of Iba1 (red) and Trem2 (green) immunostaining ( l ) and quantification ( m ) in the cortices of mice. The results showed that the humanized APOE4 knock-in significantly decreased the elevation of co-labeled cells stained by Iba1 and Trem2 antibodies in the AE-cKO mice. N = 5 mice per group. n-p. Representative images of western blotting ( n ) and quantification ( o, p ) of synaptophysin and PSD95 in the cortices of mice. The results showed that the injection of SB290157 significantly alleviated the reduction of synaptophysin ( n, p ) and PSD95 ( n, o ) in the cortices of the AE-cKO mice. N = 6 mice per group. The data represent mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, one-way ANOVA followed by Tukey’s post hoc test. Scale bars, 25 μm.

Article Snippet: The following primary antibodies were used: rat anti-Iba1 (1:500, ab283346, Abcam), goat anti-Iba1 (1:200, ab5076, Abcam), goat anti-GFAP (1:200, ab53554, Abcam), mouse anti-NeuN (1:200, MAB377, Millipore), rabbit anti-CD68 (1:200, ab283654, Abcam), sheep anti-trem2 (1:200, AF1729, R&D), rabbit anti-4G8 (1:200,800702, Bio Legend), rabbit anti-TSPO (1:2000, ab109497, Abcam), rat anti-C3 (1:200, ab11862, Abcam), and rabbit anti-C1q (1:200, ab182451,abcam).

Techniques: Injection, Immunostaining, Activation Assay, Labeling, Staining, Knock-In, Western Blot

Journal: bioRxiv

Article Title: Apolipoprotein E ε4 exacerbates microglia-mediated complement-dependent synapse loss caused by neuronal Tpk deficiency

doi: 10.1101/2025.01.16.633468

Figure Lengend Snippet: a, b. Representative images of Iba1 (green) immunostaining ( a ) and quantification ( b ) in the cortices of mice. The results showed that the humanized APOE4 knock-in significantly worsened the activation of microglia in the cortices induced by neuronal Tpk ablation, but did not affect those in the mice without Tpk knockout. c, d. Representative images of Iba1 (red) and TSPO (green) immunostaining ( c ) and quantification ( d ) in the cortices of mice. The results showed that the humanized APOE4 knock-in significantly upregulated the enhancement of co-labeled cells by Iba1 and TSPO antibodies in the cKO mice, but did not affect those in mice without Tpk knockout. e, f. Representative images of Iba1 (red) and CD68 (green) immunostaining ( e ) and quantification ( f ) in the cortices of mice. The results showed that the humanized APOE4 knock-in significantly exacerbated the elevation of co-labeled cells by Iba1 and CD68 antibodies in the cKO mice, but did affect those in mice without Tpk knockout. g, h. Representative images of Iba1 (green) and Trem2 (red) immunostaining ( g ) and quantification ( h ) in the cortices of mice. The results showed that the humanized APOE4 knock-in significantly aggravated the enhancement of co-labeled cells by Iba1 and Trem2 antibodies in the cKO mice but did not affect those in mice without Tpk knockout. N = 5 mice per group. The data represent mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, one-way ANOVA followed by Tukey’s post hoc test. Scale bars, 25 μm. i Representative images of immunostaining of Iba1 (red) and PSD95 (green) i n the cortices of mice. The results showed that microglia-mediated synaptic phagocytosis presented by PSD95 in the microglia. Scale bars, 10 μ

Article Snippet: The following primary antibodies were used: rat anti-Iba1 (1:500, ab283346, Abcam), goat anti-Iba1 (1:200, ab5076, Abcam), goat anti-GFAP (1:200, ab53554, Abcam), mouse anti-NeuN (1:200, MAB377, Millipore), rabbit anti-CD68 (1:200, ab283654, Abcam), sheep anti-trem2 (1:200, AF1729, R&D), rabbit anti-4G8 (1:200,800702, Bio Legend), rabbit anti-TSPO (1:2000, ab109497, Abcam), rat anti-C3 (1:200, ab11862, Abcam), and rabbit anti-C1q (1:200, ab182451,abcam).

Techniques: Immunostaining, Knock-In, Activation Assay, Knock-Out, Labeling

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