sheep anti-trem2  (Novus Biologicals)

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

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT enhances TREM2 recycling and Aβ endocytosis in Aβ-exposed microglia. ( A ) Flowchart of TREM2 internalization test. ( B ) After treatment with IHT, the TREM2 internalization assay was conducted in Aβ-exposed microglia, followed by the fixation of the cells and counterstaining with DAPI. Scale bar = 10 μm. ( C ) TREM2 intensity in Aβ-exposed microglia in panel B ( n > 80). ( D ) Flowchart of TREM2 recycling test. ( E ) After treatment with IHT, the TREM2 recycling assay was performed in Aβ-exposed microglia. The cells were then fixed and counterstained with DAPI. Scale bar = 10 μm. ( F ) TREM2 intensity in Aβ-exposed microglia in panel E ( n > 80). ( G ) After treatment with IHT, membrane TREM2 was labeled with anti-TREM2 antibodies in Aβ-exposed microglia and allowed to undergo internalization for 60 min. Subsequently, the cells were fixed and probed with anti-LC3 antibodies. Scale bar = 5 μm. ( H ) Colocalization ratio of TREM2 with LC3 in single cells in panel H via Manders’ colocalization coefficients ( n > 80). ( I ) After treatment with IHT, membrane TREM2 was labeled with anti-TREM2 antibodies in Aβ-exposed microglia and allowed to undergo internalization for 60 min. Subsequently, the cells were fixed and probed with anti-LAMP1 antibodies. Scale bar = 5 μm. ( J ) Colocalization ratio of TREM2 with LAMP1 in single cells in panel I via Manders’ colocalization coefficients ( n > 80. * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-way ANOVA. Nor, Normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Membrane, Labeling

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: Antibody information

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques:

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: Primer information

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Sequencing

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT downregulates TREM2 in DAM and reduces Aβ load in brains of 9-month-old APP/PS1 mice. ( A ) Escape latency of the mice to reach the target platform during the MWM training period. ( B ) The ratio of dwelling time in the target quadrant compared to all quadrants during the MWM probe test. ( C ) The escape latency of mice to reach the target quadrant during the MWM probe period. ( D ) The average swimming speed of mice during the MWM probe test ( n = 7). ( E ) Brain sections were probed with 6E10 antibodies, and microscopy images of the Aβ plaques were obtained. White arrows indicate plaques. Scale bar = 1 mm. ( F ) Brain sections were incubated with anti-TREM2 and anti-Iba1 antibodies, and microscopy images of DAM in the CA1 region were acquired. Scale bar = 10 μm. ( G ) Number of Aβ plaques in the hippocampus in panel E ( n = 6). ( H ) Number of Aβ plaques in the cortex in panel E ( n = 6). ( I ) Trem2 expression in the CA1 region was measured using qRT-PCR ( n = 3). ( J ) TREM2 intensity in the DAM in panel F (six mice in each group, with 15 cells per mouse). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test. Nor, normoxia; IHT, intermittent hypoxia training; WT, wild type; TG, APP/PS1

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Microscopy, Incubation, Expressing, Quantitative RT-PCR

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT upregulates VPS35 in DAM and Aβ-exposed microglia. ( A ) VPS35 expression in IHT-treated mice brain cortex was detected using Western blotting. (B) Grayscale values of the protein bands in panel A ( n = 6). ( C ) Vps35 expression in the CA1 regions was estimated via qRT-PCR ( n = 3). ( D and E ) Brain sections were labeled with anti-VPS35 and anti-Iba1 antibodies, and microscopy images of DAM ( D ) and microglia not associated with plaques in CA1 region were captured ( E ). Scale bar = 10 μm. ( F ) The average fluorescence intensity of VPS35 in the Iba1 + cells of DAM in panel D ( n = 7, each data point represents the average intensity in DAM of 6 plaques in each mouse brain). ( G ) VPS35 intensity in the Iba1 + cells in panel E ( n = 4). ( H ) After treatment with IHT, VPS35 expression in Aβ-exposed microglia was detected using Western blotting. ( I ) Grayscale values of the protein bands in panel H ( n = 4). ( J ) After treatment with IHT, membrane TREM2 was labeled with anti-TREM2 antibodies in Aβ-exposed microglia and allowed to undergo internalization for 30 min. Subsequently, the cells were fixed and probed with anti-VPS35 antibodies. Scale bar = 3 μm. ( K ) Colocalization ratio of TREM2 with VPS35 in single cells via Manders’ colocalization coefficients ( n > 100). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( B , C and F ) or two-way ANOVA ( G , I , and K ). n.s. indicates no significant difference. Nor, normoxia; IHT, intermittent hypoxia training; WT, wild type; TG, APP/PS1

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Labeling, Microscopy, Fluorescence, Membrane

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: VPS35 chaperone R55 augments TREM2 recycling and Aβ endocytosis by Aβ-exposed microglia. Primary microglia were co-treated with oAβ and R55. ( A ) After R55 treatment, Aβ-exposed microglia were incubated with Aβ-555 for 30 min. The cells were then fixed and probed with anti-Rab5 antibodies, followed by counterstaining using DAPI. Scale bar = 10 μm. ( B ) Colocalization ratio of Aβ-555 with Rab5 in single cells via Manders’ colocalization coefficients. ( C ) After R55 treatment, the TREM2 internalization assay was performed in Aβ-exposed microglia. Subsequently, the cells were fixed and counterstained with DAPI. Scale bar = 20 μm. ( D ) TREM2 intensity in the Aβ-exposed microglia in panel C ( n > 50). ( E ) After R55 treatment, the TREM2 recycling assay was conducted in Aβ-exposed microglia. The cells were further fixed and counterstained with DAPI. Scale bar = 20 μm. ( F ) TREM2 intensity in the Aβ-exposed microglia in panel E ( n > 80). ( G ) After R55 treatment, membrane TREM2 in Aβ-exposed microglia was labeled with anti-TREM2 antibodies and allowed to undergo internalization for 60 min. The cells were then fixed and probed with anti-LAMP1 antibodies. Scale bar = 10 μm. ( H ) Colocalization ratio of TREM2 with LAMP1 in single cells via Manders’ colocalization coefficients ( n > 100). ( I ) Vps35 expression in R55-treated Aβ-exposed microglia was estimated via qRT-PCR ( n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( H ) or two-way ANOVA ( B , D , F and I ). n.s. indicates no significant difference

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Incubation, Membrane, Labeling, Expressing, Quantitative RT-PCR

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT-induced upregulation of Aβ endocytosis by Aβ-exposed microglia depends on VPS35. Primary microglia were transfected with lentivirus expressing Cre-GFP. ( A ) Cells were labeled with anti-VPS35 antibodies. Scale bar = 20 μm. ( B ) VPS35 intensity in GFP − and GFP + cells in panel A ( n > 50). ( C ) Cells were incubated with Aβ-555 for 30 min and then fixed. Scale bar = 10 μm. ( D ) Aβ-555 intensity in GFP - or GFP + cells in panel C ( n > 80). ( E ) Cells were labeled with anti-LAMP1 and anti-TREM2 antibodies, followed by counterstaining using DAPI. Scale bar = 5 μm. ( F ) Colocalization ratio of TREM2 with LAMP1 in single cells via Manders’ colocalization coefficients ( n > 100). ( G to J ) Cells were treated with oAβ to construct Aβ-exposed microglia, followed by treatment with IHT. TREM2 internalization ( G ), TREM2 recycling ( I ), and Aβ-555 uptake ( K ) assays were conducted in the Aβ-exposed microglia (Scale bar = 20 μm). Endocytosed TREM2 ( H ), recycled TREM2 ( J ), and internalized Aβ-555 ( L ) were quantified from panels G, I , and K , respectively ( n > 50). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( B , D and F ) or two-way ANOVA ( H , J and L ). n.s. indicates no significant difference. Nor, normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Transfection, Expressing, Labeling, Incubation, Construct

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT exhibits no significant improvement in Aβ pathology in 7-months microglial VPS35-deficient APP/PS1 mice. ( A ) Labeling information for different groups. ( B ) Flowchart of the development of IHT-treated MG VPS35 KO: APP/PS1 mice. ( C ) Escape latency of the IHT-treated MG VPS35 KO: APP/PS1 mice to find the platform for the first during the MWM training period. ( D ) Swimming trajectories of the mice in the MWM probe period. ( E ) The dwelling time in the target quadrant was calculated as the percentage of the total time in the MWM probe test. ( F ) Total moving distance in the target quadrant was calculated in the MWM probe test. ( G ) The escape latency of mice to archive the target quadrant during the MWM probe period. ( H ) The average swimming speed of mice during the MWM probe test ( n = 5). ( I ) Brain sections of the IHT-treated MG VPS35 KO: APP/PS1 mice were labeled with 6E10 antibodies. White arrows indicate plaques. Scale bar = 2 mm. ( J) Brain sections of the IHT-treated MG VPS35 KO: APP/PS1 mice were probed with anti-PSD95 antibodies to label synapses in the CA1 region. Scale bar = 150 μm. ( K ) Brain sections of the IHT-treated MG VPS35 KO: APP/PS1 mice were stained with anti-Iba1 and anti-TREM2 antibodies. Scale bar = 20 μm. ( L ) Number of Aβ plaques in the hippocampus and cortex regions in panel I . ( M ) PSD95 intensity in the CA1 region in panel J . ( N ) TREM2 intensity in the DAM of CA1 region in panel K (five mice in each group, with five to eight cells per mouse). * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-way ANOVA. n.s. indicates no significant difference. Nor, normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Labeling, Staining

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT-induced attenuation of Aβ pathology and amelioration of Aβ endocytosis by DAM depends on TFEB. 9-month-old APP/PS1 mice were treated with TA1. ( A ) Brain sections of TA1-treated APP/PS1 mice were stained with anti-TFEB/anti-VPS35/anti-TREM2 and anti-Iba1 antibodies, and microscopy images of DAM in CA1 region were captured. Scale bar = 20 μm. ( B to D ) TFEB ( B ), VPS35 ( C ), and TREM2 ( D ) intensities in the DAM in panel A (six mice in each group, with 10 cells per mouse). ( E ) Sh Tfeb BV2 cells were used to construct Aβ-exposed microglia and then treated with IHT. TFEB and VPS35 expression levels were detected via Western blotting. ( F ) Grayscale values of the protein bands in panel E ( n = 3). ( G ) After treatment with IHT, sh Tfeb Aβ-exposed microglia were incubated with Aβ-555 for 30 min, followed by fixation and counterstaining with DAPI. Scale bar = 10 μm. ( H ) Aβ-555 intensity in the GFP + cells in panel G ( n > 50). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( B to D ) or two-way ANOVA ( F , H ). n.s. indicates no significant difference. Nor, normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Staining, Microscopy, Construct, Expressing, Western Blot, Incubation

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: TFEB inhibition reduces the IHT-induced upregulation of VPS35 in DAM 9-months APP/PS1 mice brains. ( A ) Brain sections of APP/PS1 mice treated with IHT or EO were probed with 6E10 antibodies, and microscopy images of the Aβ plaques were acquired. White arrows indicate plaques. Scale bar = 2 mm. ( B ) Number of Aβ plaques in the hippocampus in panel A ( n = 9). ( C to H ) Brain sections of IHT or EO-treated APP/PS1 mice were stained with anti-TFEB ( C ), anti-VPS35 ( E ), or anti-TREM2 ( G ) and anti-Iba1 antibodies, and microscopy images of DAM in CA1 region were captured. Scale bar = 20 μm. TFEB ( D ), VPS35 ( F ), and TREM2 ( H ) intensities in the DAM cells in panels C, E , and G , respectively. * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( D , F and H : four mice in each group, with three to five cells per mouse). Nor, normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Inhibition, Microscopy, Staining