Journal: iScience

Article Title: Uhrf1 governs the proliferation and differentiation of muscle satellite cells

doi: 10.1016/j.isci.2022.103928

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Myh3 , DSHB , cat# F1.652; RRID: AB_528358.

Techniques: Recombinant, Western Blot, Imaging, Multiplex Assay, Library Quantification, Methylation, Electrophoresis, Software

Journal: Cell

Article Title: The Parkinson’s disease protein alpha-synuclein is a modulator of Processing-bodies and mRNA stability

doi: 10.1016/j.cell.2022.05.008

Figure Lengend Snippet: Key resources table

Article Snippet: BCA Protein Assay Kit , Pierce , 23225.

Techniques: Western Blot, Virus, Recombinant, Protease Inhibitor, Lysis, Magnetic Beads, Membrane, Transfection, Expressing, Bicinchoninic Acid Protein Assay, Silver Staining, In Situ, Sample Prep, Luciferase, Reporter Assay, Multiplex sample analysis, Biomarker Discovery, Marker, Generated, Software, Mass Spectrometry, Imaging

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MEST promotes lung cancer invasion and metastasis by interacting with VCP to activate NF-κB signaling

doi: 10.1186/s13046-021-02107-1

Figure Lengend Snippet: SILAC quantitative proteomics characterizes MEST-regulated NF-κB pathway in lung cancer. A The scheme of two independent SILAC experiments, including forward labeling and reverse labeling. MEST-regulated proteins were identified by two independent SILAC experiments, and the differentially expressed proteins were analyzed by Ingenuity Pathway Analysis ( B ). MEST-driven functional signaling networks indicate involvement in the NF-κB signaling pathway. Red, upregulated proteins; green, downregulated proteins; white, speculative proteins. C MEST overexpression promotes nuclear translocation of NF-κB subunits (p65) in both A549 and H1299 cells. LaminB1 and GAPDH were used as markers for nucleus and cytoplasma, respectively. D A549 and H1299 overexpressing MEST were treated with either 2.5 μM of Bay11-7082 or DMSO, as indicated. Expression of NF-κB markers (including p-p65, p65, p-IκBα, and IκBα) was assessed by western blot analysis. Invasion and migration of treated cells were measured by transwell assay ( E ). F A549 and H1299 cells overexpressing MEST were treated with either 25 μM of MMP2 inhibitor I or DMSO; their invasion and migration abilities were determined by transwell assay. Scale bar, 100 μm. All data are representative of three independent experiments. Bars, S.D. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t -test)

Article Snippet: Antibodies against the following proteins were used for the experiment: VCP (#10,736–1-AP), p65 (#10,745–1-AP), Lamin B1 (#66,095–1-Ig), MYC-tag (#60,003–2-Ig), Pan-Keratin (#26,411–1-AP) and ACTB (# 66,009–1-lg) were obtained from Proteintech Group (Chicago, IL, USA); IκBα (#4814), Phospho-IκBα (Ser32/36) (#9246), phospho-p65 (Ser536; #3033), IKKα (#11,930), Phospho-IKKα/β (Ser176/180; #2697), Phospho-Stat Antibody Sampler Kit (#9914) and MMP2 (#13,132) were obtained from Cell Signaling Technology; and MEST (#ab151564) and (orb247686) were obtained from Abcam (Cambridge, MA, USA) and Biorbyt, respectively.

Techniques: Multiplex sample analysis, Quantitative Proteomics, Labeling, Functional Assay, Over Expression, Translocation Assay, Expressing, Western Blot, Migration, Transwell Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MEST promotes lung cancer invasion and metastasis by interacting with VCP to activate NF-κB signaling

doi: 10.1186/s13046-021-02107-1

Figure Lengend Snippet: MEST and VCP promote lung cancer migration and invasion via activating the NF-κB pathway. A A549-i8 and H1299-i8 cells were transfected with two anti-VCP siRNAs, and the migration and invasion abilities were determined by using a transwell assay; MMP2 and NF-κB markers (p-IκBα, IκBα, p-p65, and p65) were analyzed by western blot analysis ( B ). C MEST-overexpressing A549 and H1299 cells were transfected with si-VCP and scramble si-RNA for 24 h. Transwell assay indicates that knockdown of VCP inhibits cell migration and invasion enhanced by MEST in A549 and H1299 cells. Expression of NF-κB markers (p-IκBα, IκBα, p-p65, and p65) were analyzed by western blot analysis ( D ). E A549-i8 and H1299-i8 were transfected with VCP-HA plasmid or si-MEST for 24 h, as indicated. Transwell assay shows that knockdown of MEST in A549-i8 and H1299-i8 cells decreases cell migration and invasion, and that transfection with VCP-expression plasmid partially restores this effect. The corresponding NF-κB markers (p-IκBα, IκBα, p-p65, and p65) were analyzed by western blot analysis ( F ). G The indicated cell lines were co-transfected with MMP2 promoter-driven luciferase reporter, with pRL-TK (loading control), as well as with either the indicated plasmids or siRNA. Luciferase activity was measured ( n = 3). H Western blot analysis indicates that MMP2 expression is regulated by MEST and VCP. Bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control cells unless otherwise indicated. Scale bar, 100 μm

Article Snippet: Antibodies against the following proteins were used for the experiment: VCP (#10,736–1-AP), p65 (#10,745–1-AP), Lamin B1 (#66,095–1-Ig), MYC-tag (#60,003–2-Ig), Pan-Keratin (#26,411–1-AP) and ACTB (# 66,009–1-lg) were obtained from Proteintech Group (Chicago, IL, USA); IκBα (#4814), Phospho-IκBα (Ser32/36) (#9246), phospho-p65 (Ser536; #3033), IKKα (#11,930), Phospho-IKKα/β (Ser176/180; #2697), Phospho-Stat Antibody Sampler Kit (#9914) and MMP2 (#13,132) were obtained from Cell Signaling Technology; and MEST (#ab151564) and (orb247686) were obtained from Abcam (Cambridge, MA, USA) and Biorbyt, respectively.

Techniques: Migration, Transfection, Transwell Assay, Western Blot, Knockdown, Expressing, Plasmid Preparation, Luciferase, Control, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MEST promotes lung cancer invasion and metastasis by interacting with VCP to activate NF-κB signaling

doi: 10.1186/s13046-021-02107-1

Figure Lengend Snippet: Inhibition of VCP suppresses the pro-metastatic effect of MEST. A , B MEST-overexpressing A549 and H1299 cells were treated with either 0.5 μM of CB-5083 or DMSO for 48 h and subjected to transwell assays to detect their invasion ( A ) and migration ( B ) abilities. Scale bar, 100 μm. C The NF-κB markers including p-IκBα, IκBα, p-p65, and p65 were determined by western blot analysis. D-F NCG mice were transplanted with luciferase-labeled cells that either with or without MEST overexpression (1 × 10 6 cells per mouse) via tail vein injection ( n = 6). The indicated treatment group or control group was orally administered CB-5083 (30 mg kg −1 ) or vehicle, respectively, every 2 days. Mice were visualized 1.5 months after transplantation by using an IVIS 200 Imaging System ( D ). Lungs harvested after imaging are shown ( E ). Note that CB-5083 suppresses the metastatic nodules formed by MEST-overexpressing A549 cells in the lungs. Pulmonary metastases in the mouse model were histologically analyzed by H&E staining ( F ); scale bar, 100 μm

Article Snippet: Antibodies against the following proteins were used for the experiment: VCP (#10,736–1-AP), p65 (#10,745–1-AP), Lamin B1 (#66,095–1-Ig), MYC-tag (#60,003–2-Ig), Pan-Keratin (#26,411–1-AP) and ACTB (# 66,009–1-lg) were obtained from Proteintech Group (Chicago, IL, USA); IκBα (#4814), Phospho-IκBα (Ser32/36) (#9246), phospho-p65 (Ser536; #3033), IKKα (#11,930), Phospho-IKKα/β (Ser176/180; #2697), Phospho-Stat Antibody Sampler Kit (#9914) and MMP2 (#13,132) were obtained from Cell Signaling Technology; and MEST (#ab151564) and (orb247686) were obtained from Abcam (Cambridge, MA, USA) and Biorbyt, respectively.

Techniques: Inhibition, Migration, Western Blot, Luciferase, Labeling, Over Expression, Injection, Control, Transplantation Assay, Imaging, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MEST promotes lung cancer invasion and metastasis by interacting with VCP to activate NF-κB signaling

doi: 10.1186/s13046-021-02107-1

Figure Lengend Snippet: The expression of MEST and VCP in metastatic cancer and a schematic diagram of the action mechanism of MEST. A Two representative IHC images of MEST and VCP expression in primary lung cancer tissues and in corresponding metastatic tumor tissues. Differences in MEST and VCP expression scores between lung cancer and metastatic tumor tissues are shown ( n = 30). Scale bar, 50 μm. B Working model of MEST on promotion of cancer metastasis. MEST is upregulated in highly invasive lung cancer cells, it interacts with VCP and increases VCP to capture IκBα for degradation, leading to the nuclear translocation of p65 to activate the NF-κB pathway for cancer metastasis. In addition, MEST can induce IκBα phosphorylation via the STAT3/IKK pathway, mediating NF-κB signaling activation at multiple levels

Article Snippet: Antibodies against the following proteins were used for the experiment: VCP (#10,736–1-AP), p65 (#10,745–1-AP), Lamin B1 (#66,095–1-Ig), MYC-tag (#60,003–2-Ig), Pan-Keratin (#26,411–1-AP) and ACTB (# 66,009–1-lg) were obtained from Proteintech Group (Chicago, IL, USA); IκBα (#4814), Phospho-IκBα (Ser32/36) (#9246), phospho-p65 (Ser536; #3033), IKKα (#11,930), Phospho-IKKα/β (Ser176/180; #2697), Phospho-Stat Antibody Sampler Kit (#9914) and MMP2 (#13,132) were obtained from Cell Signaling Technology; and MEST (#ab151564) and (orb247686) were obtained from Abcam (Cambridge, MA, USA) and Biorbyt, respectively.

Techniques: Expressing, Translocation Assay, Phospho-proteomics, Activation Assay

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal IgG2a anti-FOXA2/HNF-3β , Santa Cruz , sc-101060 , IF (1:250).

Techniques: Knock-Out, Knock-In, Control, Mutagenesis, Transfection, Construct, Plasmid Preparation, Expressing, Imaging, Recombinant, Sequencing, Multiplex Assay, Software

Journal: Cell reports

Article Title: T-bet+ Memory B Cells Link to Local Cross-Reactive IgG upon Human Rhinovirus Infection

doi: 10.1016/j.celrep.2019.12.027

Figure Lengend Snippet:

Article Snippet: Metal-Antibody Conjugation Er166 (CyTOF) , Fluidigm , Cat#201166A.

Techniques: In Vitro, Blocking Assay, Multiplex Assay, Virus, Infection, Recombinant, Next-Generation Sequencing, RNA Sequencing, Software, Microscopy, Imaging, Conjugation Assay, Staining

Journal: Cell reports

Article Title: T-bet+ Memory B Cells Link to Local Cross-Reactive IgG upon Human Rhinovirus Infection

doi: 10.1016/j.celrep.2019.12.027

Figure Lengend Snippet:

Article Snippet: Metal-Antibody Conjugation Yb171 (CyTOF) , Fluidigm , Cat#201171A.

Techniques: In Vitro, Blocking Assay, Multiplex Assay, Virus, Infection, Recombinant, Next-Generation Sequencing, RNA Sequencing, Software, Microscopy, Imaging, Conjugation Assay, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Transcriptomic analysis identifies ST6GAL1 and ST3GAL3 as enriched in the cancerous ducts. A , biosynthetic pathways for glycans underlying select lectin signatures are shown. ST3GALs are responsible for transferring α-2,3-sialosides and ST6GAL1/2 for α-2,6-sialosides, and MGAT3 for bisecting GlcNAc. The glycans are annotated following the Symbolic Nomenclature for Glycans. B , transcriptomic analysis assessing the mRNA levels of select glycosyltransferases between normal adjacent and matched cancerous tissues within the same patient. C , uMAP plots representing the cells isolated from patients with PDAC (n = 24) and normal pancreata (n = 11) pooled on single-cell sequencing. The clusters representing the normal ductal cells and tumor ductal cells are highlighted. D , violin plot showing the comparison of ST6Gal1 levels in normal ( green ) versus PDAC ( blue ) ductal clusters. The clusters in each group are combined. The violin plots showing the comparison of ST6Gal1 and ST3GAL1 levels in normal ( green ) versus PDAC ( blue ) ductal clusters. The clusters in each group are combined. ns, p > 0.05; ∗ p < 0.05; student’s t test ( two-tailed ). MGAT3, beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase; ns, not statistical; PDAC, pancreatic ductal adenocarcinoma; ST3GAL3, ST3 beta-galactoside alpha-2,3-sialyltransferase 3; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; uMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Transferring, Isolation, Sequencing, Comparison, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Profiling of ST6GAL1 and SNA in human pancreatic cancer shows association with the stage and survival. A , H&E of the normal pancreas ( left ), stage I pancreatic adenocarcinoma ( center ), and stage IV PDAC ( right ) stained from a BioMax human tissue microarray. B , multiplex OPAL IF staining of SNA ( yellow ), ST6GAL1 ( red ), and DAPI ( blue ) on the corresponding normal pancreas and stage I and stage IV pancreatic adenocarcinoma purchased from BioMax human tissue microarray. The scale bars represent 25 μm. C , quantification of SNA-positive cells per high-powered field based on multiplex IF in normal versus all cancerous cases in human tissue microarray. Owing to the high number of positive cells, each HPF was given a score (1–3) based on the number of SNA-positive cells per field. D , quantification of ST6GAL1-positive cells per high-powered field in normal versus all cancerous cases in human tissue microarray. E , distribution of the total ST6GAL1-positive cells per high-powered field across normal and each stage of pancreatic cancer based on multiplex IF imaging of human pancreatic cancer tissue microarray. Statistical significance was evaluated using the Student’s t test ( two-tailed ): ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. HFP, high-powered field; ns, not statistical; PDAC, pancreatic ductal adenocarcinoma; SNA, Sambucus nigra agglutinin; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1.

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Staining, Microarray, Multiplex Assay, Imaging, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Pancreas-specific deletion of ST6GAL1 reduces disease burden in murine PDAC. A , breeding schematic illustrating the generation of novel ST6KC mice. The offspring of parental strains crossed into p48-Cre mice drive the induction of mutant KRAS G12D and deletion of ST6GAL1 under the same promoter. B , IHC staining for ST6GAL1 ( brown ) in KC and ST6KC mice (n = 3 per group). The number of ST6GAL1+ cells per high-powered field is quantified. The scale bar represents 50 μm. C , IF staining for SNA ( yellow ) and DAPI ( blue ) in KC and ST6KC mice (n = 3 per group). The number of SNA+ cells per high-powered field is quantified. The scale bar represents 100 μm. D , H&E of 14-week-old FFPE pancreata from KC and ST6KC mice (n = 5 per group). The percent of the preserved normal pancreas area is quantified per high-powered field. The scale bar represents 200 μm. E , trichrome and gomori ( blue ) stain of FFPE pancreata from 14-week-old KC and ST6KC mice (n = 5 per group). The percent of collagen deposition fibrosis is quantified per high-powered field on the right. The scale bar represents 200 μm. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student’s t test ( two-tailed ). FFPE, formalin-fixed paraffin-embedded; IF, immunofluorescence; IHC, immunohistochemical; PDAC, pancreatic ductal adenocarcinoma; SNA, Sambucus nigra agglutinin; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; ST6KC, ST6GAL1 flx/flx ;p48 Cre ; LSL KRASG12D .

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Mutagenesis, Immunohistochemistry, Staining, Two Tailed Test, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Immunohistochemical staining

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Schematic of flow sorting of CD31+CD45-BECs from mouse cortex and hippocampi. Each isolated RNA sample is a pool of BECs from 2 mouse brains. (b) FACS gating strategy to isolate single BECs. PI+ dead cells were excluded. CD11b+ and CD45+ cells were gated to exclude monocytes/macrophages and microglia. CD31+Cd11b-CD45-cells were defined as the BEC population. (c) Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of CNS cell-type specific markers. (d) FPKM values of leukocyte binding adhesion molecules including Vcam1 . (e) FPKM values of the gene transcripts in murine young and aged CD31+BECs of human plasma proteins that change with age (see for list of human plasma proteins expressed in murine BECs) (f) C57BL6 mice were injected with anti-VCAM1-DL488 or IgG isotype control (r.o.) 2 hours before perfusion to label BECs in vivo prior to brain dissociation, staining and FACS. (g) Gating and histogram plots of CD31+VCAM1+ cells sorted from the hippocampi of LPS stimulated aged (19-month old) wildtype mice injected with fluorescently tagged anti-VCAM1 mAb or IgG isotype (h) Quantification of CD31+VCAM1+cells isolated from healthy cortex (n=5 single mice/age group). **p<0.002 (i) Quantification of CD31+VCAM1+cells isolated from 4 pooled hippocampi (technical replicates shown). **p<0.005 (j) Flow gating and histogram plots of pooled (n=4 mice/ age group), young or aged hippocampi isolated from healthy mice also injected with anti-VCAM1 mAb. (k) sVCAM1 ELISA in plasma from young isochronic or heterochronic parabionts following 5 weeks of parabiosis. n=11 mice/group pooled from two independent experiments. **p<0.004, Student’s t-test . All error bars indicate SEM. (l) Confocal images in the DG of VCAM1, lectin, and Aqp4 of young isochronic or heterochronic parabionts 5 weeks after surgery. Hoechst labels cell nuclei. Scale bar = 100 μm.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Isolation, Binding Assay, Injection, In Vivo, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: (a) Heat map displaying up or down-differentially regulated genes in young versus aged BECs. There were 1006 significant differentially expressed genes (*q<0.05). (b) Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of BEC cell-type specific markers. (c) FPKM values of inflammation and activation related genes. (d) Heat map showing changes in 31 out of 74 human plasma factors with aging (p<0.05, Spearman’s correlation coefficient). Multiplex assay used (n=118 healthy humans). VCAM1 was the top factor (spearman’s correlation coefficient=0.47, p=7.7e-08). (e) Spearman correlation of VCAM1 levels and age with a significant inflection point around age 65 (Spearman’s correlation coefficient = 0.047; q< 6×10 -6 ). (f) Human sVCAM1 ELISAs in 11 young (<25 years old) or 11 aged (>65 years old) plasma from healthy donors. **p<0.005, Student’s t-test . (g) ELISA for mouse sVCAM1 in plasma from young (3-month-old; n=8), middle-aged (8–10-month-old; n=10), and aged (18-month-old; n=8) mice. ****p<0.0001, 1-way ANOVA. (h) Representative confocal images in the DG of young (3-month-old) or aged (18-month-old) mice given retro-orbital (r.o.) injections of fluorescently conjugated anti-VCAM1 and anti-Meca99 2 hours before perfusion. Hoechst labels cell nuclei. Scale bar = 50 μm. 3D rendering of the 2D images are displayed. 3D Scale bar = 50 μm. (i) Quantification of VCAM1+Lectin+ stained brain vasculature in young, middle, and aged hippocampi. n=12 young (3–4-month-old), 6 aged (18-month-old), and 6 very aged (24-month-old) mice. ****p<0.0001, 1-way ANOVA. (j) Quantification in the DG of VCAM1+Lectin+ stained brain vasculature of young isochronic or heterochronic parabionts 5 weeks after surgery. Representative Images shown in . **p<0.0006, 1-way ANOVA. n= 8-13 mice/group from two independent experiments.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Activation Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: (a) Schematic of the Blood-brain barrier (BBB). Nutrient-rich, oxygenated blood is pumped into the brain through cerebral arterioles which are protected and supported by smooth muscle cells (SMCs) that cover the endothelium and form a basement membrane layered by astrocytic end-feet of the brain parenchyma. The blood is transferred to highly specialized capillaries, which are comprised of brain endothelial cells that form unique tight junctions and are wrapped by pericytes (Peric.) within the endothelial basement membrane, which is then covered by astrocytic end-feet. BBB capillaries are the site of controlled transport of fluids and solutes into the CNS. Immuno-surveillance and occasional extravasation of leukocytes (Leuk.) into the CNS parenchyma occurs at the level of postcapillary venules, the vascular segments into which blood flows after passing through the capillaries. Postcapillary Venules contain enlarged perivascular space between the endothelial and astrocytic basement membranes where occasional immune cells can reside. , (b) Boxplot of expression levels of classical pan-endothelial and BBB-specific transcripts (c) Validation of the correlation (Spearman’s rho = 0.704) between protein and mRNA levels of 77 single BECs sorted from both Vcam1+ and Vcam1- gates. Scatterplot of Vcam1 fluorescence intensity as measured by FACs and corresponding transcript counts (per million). (d) Unbiased clustering of 112 aged and 160 young hippocampal BECs using whole transcriptome and visualization with tSNE reveals 3 molecularly distinct BEC populations. (e) tSNE visualization colored by cell identity (aged vs. young) (f) Comparison of Vcam1 expression levels in young and aged hippocampal CD31+ BECs collected from the VCAM1+ gate during FACs sorting (error bars = SD) (g) Violin plots of Vcam1 reveal differing levels of the transcript in each of the cell clusters. (h) Dotplot comparing the expression (scaled transcript counts and percent of population expressing) of various classical inflammatory, pro-angiogenic, arteriolar, venular and capillary markers between the three clusters (Cluster 0: Vcam1-negative, Cluster 1: Vcam1-high, Cluster 2: Vcam1 -low). (i) Heatmap of the scaled expression of the top 10 enriched genes (differentially expressed with p>0.05, Mann-Whitney test) in each cluster. Genes are ranked by highest log-fold change when compared to all other cells. (j) GeneAnalytics (GSEA Package)-Brain Endothelial Cell Pathway analysis of the Vcam1-high cluster. The top 11 GO pathways containing Vcam1 are highlighted here, along with the number of genes in each pathway enriched in this BEC cluster and the score assigned to the pathways. (k) GeneAnalytics (GSEA Package)-Brain Endothelial Cell Pathway analysis of the Vcam1-low cluster. The top 11 GO pathways are highlighted here, along with the number of genes in each pathway enriched in this BEC cluster and the score assigned to the pathways. (l) tSNE visualization colored by Vcam1 expression levels. Clusters are further annotated by their putative functional-phenotype and vessel segmental identity.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Membrane, Expressing, Fluorescence, Comparison, MANN-WHITNEY, Functional Assay

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Overlay of Vcam1 mRNA levels on corresponding coordinate on the Cd31 vs Vcam1 fluorescent intensity plots obtained during FACs sorting. (b) Violin plots of various inflammation-related genes in each of the 3 distinct clusters. (c) Violin plots of classical arteriolar markers in each cluster. (d) Violin plots of classical venular markers in each cluster. (e) Violin plots of various angiogenesis and Notch-signaling related genes in each of the 3 distinct clusters. (f) Volcano plots of differentially expressed genes when directly compared between Vcam1-high, low and negative clusters. Outlier genes with an adjusted p-value of >0.05 and a log2 fold change of > 1.2 are labeled for visualization.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Labeling

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Representative confocal images and quantification (b) of VCAM1, Aqp4, Lectin, with Hoechst labeling cell nuclei in the hippocampus and cerebellum of an EAE (multiple sclerosis) model. Scale bar = 100 μm. ***p<0.0007, *p<0.02. (c) Representative confocal images and quantification (d) of VCAM1, Lectin, with Hoechst labeling cell nuclei in the hippocampus and cerebellum of a Npc1-/- (Niemann Pick Disease Type C) model. Scale bar = 100 μm. ****p<0.0001, *p<0.03. (e) Representative confocal images and quantification (f) of VCAM1, Lectin, Aqp4, with Hoechst labeling cell nuclei in the Grn-/- (Frontotemporal Dementia) model. Scale bar = 100 μm.***p<0.0005. Student’s t-test . All error bars indicate SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Labeling

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Vcam1 fl/fl Slco1c1-Cre ERT2+/- (Cre+) or Cre ERT2-/- (Cre-) littermates (3-month-old) were treated daily with tamoxifen (i.p. 150 mg/kg) for 5 days followed by 4 days of rest. Mice received 3 LPS injections (0.5 mg/kg i.p.) at 28 hours, 22 hours, and 2 hours prior to perfusion. Mice also received a retro-orbital injection of fluorescently conjugated mouse anti-VCAM1 mAb (100μg) 2 hours prior to perfusion. Representative confocal images of cortex and DG for VCAM1 and Hoechst to label cell nuclei. Loss of Vcam1 in Cre+ mice, but not Cre-, in BBB endothelium, but not in meninges is shown. Scale bar = 100 μm. (b) FACS gating strategy to isolate single BECs. PI+ dead cells were excluded. CD11a/b, CD45, and Ter-119 negative cells were gated to exclude erythrocytes, monocytes/macrophages and microglia. CD13 and ACSA-2 staining was applied to exclude pericytes and astrocytes, respectively. CD31+MECA99+ cells were defined the BEC population. (c) Quantification of (d) flow cytometry that was performed on primary BECs isolated from Cre+ or Cre-mice treated as described in (a). n=3 Cre+ or Cre-mice received LPS, while one Cre-mouse was given PBS vehicle control instead. The VCAM1 gate was set based on a Cre-mice injected with fluorescently conjugated IgG. ***p<0.0007; Unpaired Student’s t-test ; Error bar represents SEM. (e) Experimental Design. n= 6-7 mice/group. (f) Quantification of VCAM1+ percent area in lectin+ vasculature of immunostained sections from 5–6 mice/group. ****p<0.0007; Student’s t-test . (g-i) Quantification of the total number of BrdU+ cells, BrdU+Sox2+ co-labeled neural progenitor cells, and DCX+ immature neurons in the DG of immunostained sections. **p<0.003, Student’s t-test . (j-m) Quantification of Iba1 and CD68 in the DG of immunostained sections. All error bars represent SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Injection, Staining, Flow Cytometry, Isolation, Labeling

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to and (a) Representative images of Bend.3 cells immunostained for BBB specific markers of adherens junctions (AJ) and tight junctions (TJ), specifically ß-catenin, Claudin-5, and VE-Cadherin. Hoechst labels cell nuclei. Scale bar = 100 μm. (b) Dose response graph depicting cultured Bend.3 cells stimulated overnight with increasing concentrations of recombinant mouse TNF-α followed by flow cytometry to quantify %CD31 + VCAM1+ cells. n=2 pooled samples per condition. (c) Primary BECs and Bend.3 cells cultured in 10% young or aged mouse plasma (YMP: 3-month old; AMP: 18-month-old) or young or aged human plasma (<25 years or >65 years, YHP/AHP) for 16 hours then stained for VE-Cadherin, VCAM1, and Hoechst to label cell nuclei. Representative images are shown. Scale bar = 100 μm. (d) Quantification of VCAM1 %area staining. *p<0.04, ***p<0.0004, ****p<0.0001 n=4-6/group, student’s t-test . All error bars indicate SEM. (e) Bend.3 cells cultured in 10% young or aged mouse plasma (YMP/AMP) for 16 hours followed by flow cytometry of CD31 and VCAM1. Graph of %CD31+VCAM1+ quantification shown with histogram and flow cytometry gating of Bend.3 cells. **p<0.005, student’s t-test . (f) Quantification of %CD31+ cells colabeled with ICAM1, E-Selectin, or P-Selectin. n=6 replicates/group. All error bars indicate SEM. Histogram plots shown in (g). (h) Representative images of ICAM1, Meca99, lectin, and Hoechst to label cell nuclei of young (3-month-old) mice which received 7 r.o. injections of young (3 mo) or aged (18 mo) pooled plasma over 4 days as described in schematic. Quantification on the right. Scale bar = 100 μm.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Cell Culture, Recombinant, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: (a) Schematic of experimental design. n=10-11 mice/group. (b) Representative confocal images (left) and quantification (right) of VCAM1+lectin+ in the DG. Hoechst labels cell nuclei. Arrows indicate VCAM1+ vessels. Scale bar = 100 μm. ****p<0.0002. (c) Top: Gating and histogram plots of CD31+VCAM1+ cells isolated from LPS stimulated young (3-month old) wildtype mice injected with fluorescently tagged anti-VCAM1 mAb or IgG isotype control (r.o.) 2 hours before sacrifice. Bottom: Flow gating and histogram plots of pooled (n=4 mice/plasma treatment), young or aged hippocampi isolated from plasma-injected young mice administered anti-VCAM1 mAb prior to sacrifice. Quantification (d) of CD31+VCAM1+cells isolated from healthy cortex (n=4 single mice/plasma treatment) and of 4 technical replicates of pooled hippocampi. **p<0.004., *p<0.02. (e) Representative confocal images and quantification (f) in the DG of BrdU+, Sox2+, and GFAP. Scale bar = 100 μm. Purple lines outline the SGZ and arrows indicate proliferating neural precursor cells. **p<0.01, *p<0.03. (g) Representative confocal images and quantification (h) in the DG of DCX (white). Scale bar = 100 μm. ***p<0.0002. (i) Representative confocal images and quantification (j) in the DG of CD68, Iba1, and Hoechst. Scale bar = 100 μm. **p<0.005, *p<0.04 Student’s t-test . All error bars indicate SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Isolation, Injection

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Experimental design. n=5 mice/group (b) Quantification of %VCAM1+Lectin+ staining. *p<0.03. (c) Representative confocal images and quantification (d) in the DG of EdU+ proliferating cells and EdU+ and Sox2+ colabeled proliferating neural progenitor cells. GFAP labels astrocytes and neural stem cells. Scale bar = 50 μm. *p<0.05. (e) Representative confocal images and (f) quantification in the DG of CD68, Iba1 and Hoechst to label cell nuclei. Scale bar = 100 μm. p=0.08; **p<0.05. Student’s t-test . All error bars represent SEM. (g) Schematic. n= 6-7 mice/group. (h) Representative confocal images and (i) quantification in the DG of VCAM1 (arrows). Hoechst labels cell nuclei. *p<0.05. Student’s t-test . Scale bar = 100 μm. (j) Quantification in the DG of BrdU+ and Sox2+ neural precursor cells and triple labeled GFAP+ neural stem cells from confocal images of immunostained sections. Scale bar = 100 μm. *p<0.02, **p<0.05. (k) Representative confocal images and quantification (l) in the DG of Iba1, CD68, and Hoechst to label cell nuclei. Scale bar = 100 μm. **p<0.05. Student’s t-test . Error bars represent SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Staining, Labeling

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Representative 2D and 3D Z-stacked high magnification confocal images (51 slices with an interval of 0.4 um) of VCAM1 in the granular layer of the DG of the hippocampus of a young (3-month-old) NSG mouse acutely treated with Aged Human Plasma (AHP). Brain sections were co-stained with DCX and NeuN to label immature and mature granule neurons, respectively. VCAM1 is not expressed in these cell types. Light blue lines outline the granule layer. 2D Scale bar = 50 μm. Two 3D renderings of the 2D images are displayed with 180°rotations. 3D Scale bar = 20 μm. (b) Representative 2D and 3D Z-stacked high magnification confocal images (51 slices with an interval of 0.4 um) of VCAM1 in the granular layer of the DG of the hippocampus co-stained with Sox2 and GFAP to label neural stem and progenitor cells (Sox2+GFAP+) and hilur GFAP+ astrocytes. VCAM1 is not expressed in these cell types in the DG. Light blue lines outline the granule layer. 2D Scale bar = 50 μm. Two 3D renderings of the 2D images are displayed with 180°rotation. 3D Scale bar = 20 μm.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Staining

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: (a) Experimental design. n=7-8 mice/group. (b) Representative confocal images in the DG of VCAM1, MECA-99, and Aqp4. Hoechst labels cell nuclei. Scale bar = 200 pm for merged images and scale bar= 100 pm for the zoomed VCAM1 and MECA-99 merged images outlined with white squares. (c) Quantification of VCAM1+ lectin+ vasculature ***p<0.004. (d) Mouse sVCAM1 ELISA of plasma samples. *p<0.03. (e) BrdU quantification and representative confocal images (f) and BrdU+Sox2+ quantification (g) in the DG of brain sections immunostained for BrdU and Sox2. White dotted lines outline the SGZ; Scale bar = 200 μm. *p<0.03, **p<0.02. (h) DCX+ quantification and representative confocal images (i) in the DG. Hoechst labels cell nuclei. Scale bar = 100 μm. **p<0.002. (j) Representative confocal images and quantification (k) from the DG of CD68 and Iba1. Hoechst labels cell nuclei. Scale bar = 100 μm. ***p<0.0009, **p<0.007, 2-way ANOVA with Tukey’s post-hoc test. All error bars represent SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Brain endothelial and epithelial-specific Vcam1 deletion in young mice mitigates the negative effects of 3 weeks of aged plasma administration. Related to (a) Experimental Design. n=8 mice/group. (b) Representative confocal images and (c) quantification in the DG of VCAM1, MECA-99, and Aqp4. Hoechst labels cell nuclei. Scale bar = 100 μm. Arrows point to VCAM1+ vessels. (4 mice/group analyzed). ***p<0.0009. (d-f) Quantification of the total number of BrdU+ cells, BrdU+Sox2+ neural progenitor cells, and DCX+ immature neurons in the DG of immunostained sections. *p<0.02, **p<0.007. (g-h) Quantification of Iba1 and CD68 in the DG of immunostained sections. ****p<0.0001, 2-way ANOVA with Tukey’s post-hoc test. All error bars represent SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques:

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Experimental Design. n=6 isochronic young pairs (3-month-old C57BL6/J mice and 3.5-month-old Cre+ mice) and 9 heterochronic pairs (18-month-old C57BL6 mice and 3.5-month-old Cre+ mice). (b) Quantification of VCAM1+ percent area in lectin+ vasculature of immunostained sections. **p<0.0001. (c-d) Quantification of the total number of BrdU+Sox2+ neural progenitor cells and DCX+ immature neurons in the DG of immunostained sections. ****p<0.0001. (e) Quantification of the total number of EdU+NeuN+ mature neurons in the DG of immunostained sections. ****p<0.0001. (f-g) Quantification of Iba1 and CD68 in the DG of immunostained sections. ***p<0.0001. 1-way ANOVA with Tukey’s post-hoc test. All error bars represent SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques:

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: (a) Experimental design. n=7-8 mice/group. (b) Ponceau S stain showing total protein pull-down from plasma by both IgG and anti-VCAM1 mAb conjugated beads. (c) Western blot showing human sVCAMI (93 kDa) pulled down during immunodepletion. (d) Human sVCAMI ELISA of depleted plasma. (e) Representative confocal images and quantification (f) in the DG of VCAM1, MECA-99, and Aqp4. Hoechst labels cell nuclei. Scale bar = 50 pm for merged images and scale bar= 20 pm for the 4x zoomed single channel VCAM1 images outlined with white squares. Arrows indicate VCAM1+ vessels. n=5 mice/group. ***p<0.001. (g) Quantification of the total number of BrdU+ and BrdU+Sox2+ co-labeled neural progenitor cells in the DG of immunostained sections. *p<0.009. (h) Quantification and representative confocal images (i) of the DG for DCX and Hoechst to label cell nuclei. Scale bar = 100 μm. n= 5 mice/group **p<0.003. (j) Quantification of the Iba1+ and CD68+ staining from confocal images in the DG. *p<0.05, **p<0.01, ***p<0.004. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. All error bars represent SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: (a) Experimental design. n=10 mice/group. (b) Representative confocal images and quantification (e) (n= 5 mice/group) in the DG of VCAM1, lectin, and Aqp4. Hoechst labels cell nuclei. White arrows point to VCAM1+ vessels. Scale bar = 100 μm. ****p<0.0001, 2-way ANOVA. (c) Representative confocal images and quantification (f) in the DG of EdU and Sox2. Hoechst labels cell nuclei. Arrows indicate proliferating neural precursor cells. The SGZ is outlined with white lines. Scale bar = 50 μm. *p<0.02, 2-way ANOVA. (d) Representative confocal images and quantification (g) in the DG of CD68 and Iba1. Hoechst labels cell nuclei. Scale bar = 100 μm. ****p<0.0007, ***p<0.002, *p<0.04, 2-way ANOVA.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques:

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to - (a) Schematic. n=10, 3-month-old NSG mice per group. (b) Quantification and (c) representative confocal images in the DG of VCAM1 (arrows), lectin, and Aqp4. Hoechst labels cell nuclei. Scale bar = 100 μm. **p<0.009. (d) Quantification in the DG of CD68 in Iba1+ stained microglia. ***p<0.0007, 1-way ANOVA with Tukey’s post-hoc test. Error bars represent SEM. (e) Representative confocal images in the DG of BrdU+, Sox2+ and GFAP+ neural precursor cells. Boxed areas in the SGZ of images in low magnification (scale bar = 100 pm) are shown in right panels in high magnification (scale bar = 50 pm). Quantification of BrdU+Sox2+ progenitor cells (f) and DCX+ immature neurons (g) from confocal images. *p<0.02. (h) sVCAM1 ELISA of the plasma of young (4-month-old) and aged (19-month-old) female mice. ****p<0.0001. (i) Schematic. Aged (18-month-old) C57BL6/J female mice received i.p. injections of a mouse specific anti-VCAM1 mAb or IgG isotype control (9 mg/kg) every 3 days for a total of 7 injections. Mice also received BrdU daily (100 mg/kg i.p.) for 6 consecutive days followed by perfusion 2 days after the last injection. n=9–10 mice/group. (j) Quantification of VCAM1+Lectin+ staining from confocal images in the DG. *p<0.02, 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. (k) Quantification of BrdU+ and BrdU+Sox2+ staining from confocal images in the DG. ***p<0.0004, *p<0.04. (l) Quantification of Iba1 and CD68 staining from confocal images in the DG. **p<0.001, *p<0.05, Student’s t-test unless otherwise noted. All error bars represent SEM.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Injection

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: (a) Experimental design for anti-VCAM1. n=7 mice/group. (b) Representative confocal images and quantification (d) in the DG of BrdU and Sox2. Arrows indicate proliferating neural precursor cells. The white lines outline the SGZ. Scale bar = 100 μm. *p<0.04, **p<0.003, Student’s t-test . (c) Representative confocal images and quantification (e) in the DG of CD68, Iba1, and Hoechst. Scale bar = 100 μm. p***<0006, **p<0.02, *p<0.04, Student’s t-test . All error bars represent SEM. (f) Experimental design for anti-VLA-4. n=7 mice/group. (g) Representative confocal images and quantification (I) of VCAM1, Lectin, and Hoechst to label cell nuclei. Scale bar = 100 μm. (h) Representative confocal images and quantification (k) in the DG of CD68, Iba1, and Hoechst. Scale bar = 100 μm. *p<0.02, 1-way ANOVA. All error bars represent SEM. (j) Quantification of confocal images of the DG of NPCs co-labeled with BrdU and Sox2.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Labeling

Journal: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: In young healthy mice, neurovascular homeostasis is maintained with low expression levels of systemic and BBB-specific VCAM1, active neurogenesis and nonreactive microglia in a low inflammation environment. During aging or exposure to aged plasma, we propose: 1) Factors in aged plasma induce BEC activation and upregulation of VCAM1. 2) VCAM1 facilitates tethering, but not transmigration, of leukocytes which sustain BEC inflammation. 3) Inflamed VCAM1+ brain endothelium relay (unknown) signals to the parenchyma leading to a loss of homeostasis, decline in neurogenesis and chronic activation of microglia.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Expressing, Activation Assay, Transmigration Assay