Journal: Neuron

Article Title: TREM2 is a receptor for β-amyloid which mediates microglial function

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: (A and B) The binding of Aβ1–42 monomers or oligomers with Fc, TREM2-Fc or TREM1-Fc. All values were normalized to input. M: marker. n=5 independent experiments, ***P<.001 by unpaired t-test.

Article Snippet: Rabbit anti-human TREM2 , Cell Signaling Technology , Cat# 91068 RRID:AB_2721119.

Techniques: Binding Assay, Marker

Journal: Neuron

Article Title: TREM2 is a receptor for β-amyloid which mediates microglial function

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: (A–D) TREM2 deficiency attenuates microglia-mediated Aβ degradation. Cultured microglia from WT or TREM2 KO mice were incubated with 200 nM FAM-Aβ1–42 aggregates at 37°C for as the time indicated in (A), and washed/re-incubated in Aβ-free media with or without MG132 (B–D) or chloroquine (CQ) (C and D). FAM-Aβ in WT or TREM2 KO microglia at different time points was determined by flow cytometry (A and B), or confocal microscopy (C). All values were normalized to WT FAM-Aβ levels 2h after uptake. Quantification of FAM-Aβ fluorescence is shown in (D). Scale bar=20 µm. n= 3 independent measurements, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Tukey post hoc test.

Article Snippet: Rabbit anti-human TREM2 , Cell Signaling Technology , Cat# 91068 RRID:AB_2721119.

Techniques: Cell Culture, Incubation, Flow Cytometry, Confocal Microscopy, Fluorescence

Journal: Neuron

Article Title: TREM2 is a receptor for β-amyloid which mediates microglial function

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: (A) IL-6, MIP-1α, or Arg1 expression was determined by qRT-PCR in WT or TREM2 KO microglia in the presence or absence of 1 µM Aβ. n = 3 independent experiments, *P < 0.05, **P < 0.01, by two-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Rabbit anti-human TREM2 , Cell Signaling Technology , Cat# 91068 RRID:AB_2721119.

Techniques: Expressing, Quantitative RT-PCR

Journal: Neuron

Article Title: TREM2 is a receptor for β-amyloid which mediates microglial function

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: (A–C) Analysis of WT or TREM2 KO brain at 16 hours after FAM-Aβ injection. n = 8 for WT, n = 9 for KO mice.

Article Snippet: Rabbit anti-human TREM2 , Cell Signaling Technology , Cat# 91068 RRID:AB_2721119.

Techniques: Injection

Journal: Neuron

Article Title: TREM2 is a receptor for β-amyloid which mediates microglial function

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet:

Article Snippet: Rabbit anti-human TREM2 , Cell Signaling Technology , Cat# 91068 RRID:AB_2721119.

Techniques:

Journal: Neuron

Article Title: TREM2 is a receptor for β-amyloid which mediates microglial function

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-human TREM2 , Cell Signaling Technology , Cat# 91068 RRID:AB_2721119.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Mutagenesis, Plasmid Preparation, Software

Journal: Science Advances

Article Title: TMEM106B regulates microglial proliferation and survival in response to demyelination

doi: 10.1126/sciadv.add2676

Figure Lengend Snippet: ( A and B ) Brain sections from WT and KO mice untreated or treated with CPZ for 5 weeks were stained with IBA1 and CD68 antibodies. Representative images in the CC region were shown (A). Scale bars, 100 μm. CD68 levels per microglia and the number of CD68 + IBA1 + microglia were quantified (B). Data represent the means ± SEM. Statistical significance was analyzed by two-way ANOVA ( n = 3 to 4 mice per group). ( C and D ) Brain sections from WT and KO mice untreated or treated with CPZ for 5 weeks were stained with Galectin-3, TREM2, and IBA1 antibodies. Representative images in CC were shown (C). Scale bars, 10 μm. Galectin-3 and TREM2 levels per microglia were quantified (D). Data represent the means ± SEM. Statistical significance was analyzed by unpaired two-tailed Student’s t test ( n = 4 mice per group). ( E and F ) Western blot analysis of IBA1 and Galectin3 in the cortical lysates from WT and KO mice fed with CPZ for 5 weeks. The protein levels were quantified and normalized to GAPDH. Data represent the means ± SEM. Statistical significance was analyzed by unpaired two-tailed Student’s t test ( n = 3 to 4 mice per group). ( G ) Microglia morphology analysis at the different time points was done using a ramification index [RI = 4π × cell area/(cell perimeter) 2 ] that describes microglial cell shape. Ten microglia were analyzed for mice fed with a normal diet. A total of 40 to 70 cells per group from three to five independent mice per group at different time points were analyzed. Data represent the means ± SEM. Statistical significance was analyzed by unpaired two-tailed Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following antibodies were used in this study: mouse anti-ACBD3 (Santa Cruz Biotechnology, sc-101277), mouse anti-APC (Millipore, OP80), goat anti-CathB (R&D Systems, AF965), goat anti-CathD (R&D Systems, AF1029), rat anti-CD68 (Bio-Rad, MCA1957), rabbit anti–cleaved caspase 3 (Cell Signaling Technology, 9661), mouse anti–galectin-3 (BioLegend, 126702), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Proteintech Group, 60004-1-Ig), mouse anti-GFAP (GA5) (Cell Signaling Technology, 3670S), rat anti-mouse LAMP1 (BD Biosciences, 553792), rabbit anti-IBA1 (Wako, 01919741), goat anti–AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-Ki67 (Invitrogen, 14-5698-82), mouse anti-PCNA (Cell Signaling Technology, 2586), mouse anti-MBP (Millipore, SMI-99), goat anti-OLIG2 (R&D Systems, AF2418), mouse anti-PLP (Millipore, MAB388), rat anti-TREM2 clone 5F4 (Millipore, MABN2321), rat anti-TREM2 (R&D Systems, MAB17293), goat anti-hTREM2 (R&D Systems, AF1828), and sheep anti-TREM2 (R&D Systems, AF1729).

Techniques: Staining, Two Tailed Test, Western Blot

Journal: Science Advances

Article Title: TMEM106B regulates microglial proliferation and survival in response to demyelination

doi: 10.1126/sciadv.add2676

Figure Lengend Snippet: ( A to C ) Four-month-old WT and KO mice were untreated or treated with LPS intracerebroventricularly. Brains were harvested 72 hours after injection, and brain sections were stained with IBA1 and CD68 antibodies. Representative images from the cortex and dentate gyrus of the hippocampus were shown (A). Scale bar, 100 μm. The number of IBA1 + microglia (B) and CD68 levels per microglia (C) were quantified in the cortex and hippocampus for the experiment. Data represent the means ± SEM. Statistical significance was analyzed using two-way ANOVA ( n = 3 to 4 mice per group). ** P < 0.01 and *** P < 0.001. ( D to F ) Brain sections from LPS-treated WT and KO mice were stained with Ki67, TREM2, and IBA1 antibodies. Representative images from the cortex and dentate gyrus of the hippocampus were shown (D). Scale bar, 10 μm. The number of Ki67 + microglia (E) and TREM2 levels per microglia (F) in the cortex and hippocampus region were quantified. Data represent the means ± SEM. Statistical significance was calculated using the unpaired two-tailed Student’s t t est ( n = 3 mice per group). * P < 0.05; ** P < 0.01. ( G and H ) Brain sections from untreated or LPS-treated WT and KO mice were stained with MBP antibodies. Representative images from the cortex and dentate gyrus of the hippocampus were shown (G). Scale bars, 100 μm. MBP intensities were quantified in (H). Data represent the means ± SEM. Statistical significance was analyzed by two-way ANOVA ( n = 3 to 4 mice per group). * P < 0.05.

Article Snippet: The following antibodies were used in this study: mouse anti-ACBD3 (Santa Cruz Biotechnology, sc-101277), mouse anti-APC (Millipore, OP80), goat anti-CathB (R&D Systems, AF965), goat anti-CathD (R&D Systems, AF1029), rat anti-CD68 (Bio-Rad, MCA1957), rabbit anti–cleaved caspase 3 (Cell Signaling Technology, 9661), mouse anti–galectin-3 (BioLegend, 126702), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Proteintech Group, 60004-1-Ig), mouse anti-GFAP (GA5) (Cell Signaling Technology, 3670S), rat anti-mouse LAMP1 (BD Biosciences, 553792), rabbit anti-IBA1 (Wako, 01919741), goat anti–AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-Ki67 (Invitrogen, 14-5698-82), mouse anti-PCNA (Cell Signaling Technology, 2586), mouse anti-MBP (Millipore, SMI-99), goat anti-OLIG2 (R&D Systems, AF2418), mouse anti-PLP (Millipore, MAB388), rat anti-TREM2 clone 5F4 (Millipore, MABN2321), rat anti-TREM2 (R&D Systems, MAB17293), goat anti-hTREM2 (R&D Systems, AF1828), and sheep anti-TREM2 (R&D Systems, AF1729).

Techniques: Injection, Staining, Two Tailed Test

Journal: Science Advances

Article Title: TMEM106B regulates microglial proliferation and survival in response to demyelination

doi: 10.1126/sciadv.add2676

Figure Lengend Snippet: ( A to C ) Cx3cr1 +/CreER Tmem106b flox/flox mice and Cx3cr1 +/CreER mice were fed with tamoxifen for 2 weeks at 4 weeks of age and untreated or treated with LPS intracerebroventricularly at 12 weeks of age. Brains were harvested 72 hours after injection, and brain sections were stained with IBA1 and CD68 antibodies. Representative images from the cortex and dentate gyrus of the hippocampus were shown (A). Scale bars, 100 μm. The number of IBA1 + microglia was quantified (B). Data represent the means ± SEM. Statistical significance was analyzed using two-way ANOVA ( n = 3 to 4 mice per group). (C) Brain sections from experiments described in (A) were stained with Ki67 and IBA1 antibodies, and the percentage of Ki67 + IBA1 + microglia in the cortex and hippocampus region after LPS treatment was quantified. Data represent the means ± SEM; unpaired two-tailed Student’s t test ( n = 3 to 4 mice per group). ( D and E ) Brain sections from LPS-treated control and 106b fl/fl mice were stained with CD68, TREM2, and IBA1 antibodies. Representative images from the cortex and dentate gyrus of the hippocampus were shown (D). Scale bars, 10 μm. The levels of CD68 and TREM2 per microglia were quantified (E). Data represent the means ± SEM; unpaired two-tailed Student’s t test ( n = 3 mice per group). ( F and G ) Brain sections from untreated or LPS-treated control and 106b fl/fl mice were stained with MBP antibodies. Representative images from the cortex and dentate gyrus of the hippocampus were shown (F). Scale bars, 100 μm. MBP intensities were quantified in (G). Data represent the means ± SEM; two-way ANOVA ( n = 3 to 4 mice per group). * P < 0.05 and ** P < 0.01.

Article Snippet: The following antibodies were used in this study: mouse anti-ACBD3 (Santa Cruz Biotechnology, sc-101277), mouse anti-APC (Millipore, OP80), goat anti-CathB (R&D Systems, AF965), goat anti-CathD (R&D Systems, AF1029), rat anti-CD68 (Bio-Rad, MCA1957), rabbit anti–cleaved caspase 3 (Cell Signaling Technology, 9661), mouse anti–galectin-3 (BioLegend, 126702), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Proteintech Group, 60004-1-Ig), mouse anti-GFAP (GA5) (Cell Signaling Technology, 3670S), rat anti-mouse LAMP1 (BD Biosciences, 553792), rabbit anti-IBA1 (Wako, 01919741), goat anti–AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-Ki67 (Invitrogen, 14-5698-82), mouse anti-PCNA (Cell Signaling Technology, 2586), mouse anti-MBP (Millipore, SMI-99), goat anti-OLIG2 (R&D Systems, AF2418), mouse anti-PLP (Millipore, MAB388), rat anti-TREM2 clone 5F4 (Millipore, MABN2321), rat anti-TREM2 (R&D Systems, MAB17293), goat anti-hTREM2 (R&D Systems, AF1828), and sheep anti-TREM2 (R&D Systems, AF1729).

Techniques: Injection, Staining, Two Tailed Test

Journal: Science Advances

Article Title: TMEM106B regulates microglial proliferation and survival in response to demyelination

doi: 10.1126/sciadv.add2676

Figure Lengend Snippet: ( A and B ) Fixed (A) or live (B) WT and KO primary microglia were stained with the indicated antibodies. Scale bar, 10 μm. TREM2 levels per cell were quantified for a total of 142 WT microglia and 122 KO microglia from four independent batches of microglial culture (A) or a total of 86 WT microglia and 74 KO microglia from three independent batches of microglial culture (B). Data represent the means ± SEM. Statistical significance was analyzed by unpaired two-tailed Student’s t test [ n = 4 (A) or n = 3 (B)]. ** P < 0.01. ( C ) Western blot analysis of TREM2 levels in the microglia cultured from WT and KO mice. The protein levels were quantified and normalized to GAPDH. Data represent the means ± SEM. Statistical significance was analyzed by unpaired two-tailed Student’s t test ( n = 3 independent batches of microglial culture). * P < 0.05. Asterisk indicated the nonspecific band. ( D ) HeLa cells stably expressing TREM2 were transfected with FLAG-tagged TMEM106B or control vector and stained with TREM2, ACBD3, and TMEM106B antibodies. Scale bar, 10 μm. TREM2 levels were quantified and normalized to vector control. Data represent the means ± SEM. Statistical significance was analyzed by one-sample t test ( n = 3 independent experiments). * P < 0.05. ( E to G ) Myc-tagged TREM2 were cotransfected with GFP-tagged TMEM106B, GFP-tagged CD68, or GFP-expressing constructs (E) or cotransfected with FLAG-tagged TMEM106B or empty vector (F) into HEK293T cells as indicated. Untagged TMEM106B was cotransfected with Myc-TREM2 or empty vector into HEK293T cells as indicated (G). Cells were harvested 48 hours after transfection, and lysates were subjected to immunoprecipitations with anti-GFP (E), anti-FLAG (F), or anti-Myc (G) antibodies. IP products were analyzed by Western blots using the indicated antibodies.

Article Snippet: The following antibodies were used in this study: mouse anti-ACBD3 (Santa Cruz Biotechnology, sc-101277), mouse anti-APC (Millipore, OP80), goat anti-CathB (R&D Systems, AF965), goat anti-CathD (R&D Systems, AF1029), rat anti-CD68 (Bio-Rad, MCA1957), rabbit anti–cleaved caspase 3 (Cell Signaling Technology, 9661), mouse anti–galectin-3 (BioLegend, 126702), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Proteintech Group, 60004-1-Ig), mouse anti-GFAP (GA5) (Cell Signaling Technology, 3670S), rat anti-mouse LAMP1 (BD Biosciences, 553792), rabbit anti-IBA1 (Wako, 01919741), goat anti–AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-Ki67 (Invitrogen, 14-5698-82), mouse anti-PCNA (Cell Signaling Technology, 2586), mouse anti-MBP (Millipore, SMI-99), goat anti-OLIG2 (R&D Systems, AF2418), mouse anti-PLP (Millipore, MAB388), rat anti-TREM2 clone 5F4 (Millipore, MABN2321), rat anti-TREM2 (R&D Systems, MAB17293), goat anti-hTREM2 (R&D Systems, AF1828), and sheep anti-TREM2 (R&D Systems, AF1729).

Techniques: Staining, Two Tailed Test, Western Blot, Cell Culture, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Construct

Journal: Science Advances

Article Title: TMEM106B regulates microglial proliferation and survival in response to demyelination

doi: 10.1126/sciadv.add2676

Figure Lengend Snippet: ( A to C ) Corpus callosum sections of postmortem human brain tissues (table S1) were stained with MBP and IBA1 antibodies. Scale bar, 100 μm. Five to ten 20× images were taken from each section. IBA1-positive microglia number per image and MBP levels were quantified in (B) and (C), respectively. MBP intensity was normalized to the average of AA samples. Data represent the means ± SEM. Statistical significance was analyzed by unpaired two-tailed Student’s t test ( n = 6 to 7 for each genotype). * P < 0.05. ( D ) A model to illustrate the function of TMEM106B in microglia and myelination: Dysfunction of TMEM106B in microglia leads to decreased levels of TREM2, lysosomal abnormalities, and other defects; these alterations result in reduced microglia survival and proliferation in response to myelin debris, contributing to myelination deficits.

Article Snippet: The following antibodies were used in this study: mouse anti-ACBD3 (Santa Cruz Biotechnology, sc-101277), mouse anti-APC (Millipore, OP80), goat anti-CathB (R&D Systems, AF965), goat anti-CathD (R&D Systems, AF1029), rat anti-CD68 (Bio-Rad, MCA1957), rabbit anti–cleaved caspase 3 (Cell Signaling Technology, 9661), mouse anti–galectin-3 (BioLegend, 126702), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Proteintech Group, 60004-1-Ig), mouse anti-GFAP (GA5) (Cell Signaling Technology, 3670S), rat anti-mouse LAMP1 (BD Biosciences, 553792), rabbit anti-IBA1 (Wako, 01919741), goat anti–AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-Ki67 (Invitrogen, 14-5698-82), mouse anti-PCNA (Cell Signaling Technology, 2586), mouse anti-MBP (Millipore, SMI-99), goat anti-OLIG2 (R&D Systems, AF2418), mouse anti-PLP (Millipore, MAB388), rat anti-TREM2 clone 5F4 (Millipore, MABN2321), rat anti-TREM2 (R&D Systems, MAB17293), goat anti-hTREM2 (R&D Systems, AF1828), and sheep anti-TREM2 (R&D Systems, AF1729).

Techniques: Staining, Two Tailed Test