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: (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: (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: 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: 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: (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: (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: 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: 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: (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: (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: 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