Journal: Developmental cell

Article Title: Aging suppresses sphingosine-1-phosphate (S1P) chaperone ApoM in circulation resulting in maladaptive organ repair

doi: 10.1016/j.devcel.2020.05.024

Figure Lengend Snippet: Key Resources Table

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Experimental Models: Organisms/Strains Mouse: Floxed S1pr1 ( Allende et al., 2003 ) RRID: MGI:2681963 Mouse: Apom transgenic ( Christoffersen et al., 2008 ) PMID: 18006500 Mouse: Apom knockout ( Christoffersen et al., 2008 ) PMID: 18006500 Mouse:Cdh5-Cre ERT2 ( Wang et al., 2010b ) RRID: MGI:3848984 Mouse: Albumin-Cre The Jackson Laboratory RRID: IMSR_JAX: 003574 Mouse: S1pr1-Gfp ( Kono et al., 2014 ) PMID: 24667638 Mouse: C57BL6/J The Jackson Laboratory N/A Bacterial and Viral Strains AAV9 ( Gao et al., 2004 ) PMID:15163731 Antibodies Rabbit polyclonal anti-Collagen I Abcam Cat#ab34710 Rabbit polyclonal anti-SMA Abcam Cat#ab5694 Rabbit polyclonal anti-desmin Abcam Cat#ab15200 Goat polyclonal anti-VE-Cadherin R&D Cat#AF1002 Rabbit polyclonal anti-SFTPC Abcam Cat#ab28744 Rabbit polyclonal anti-Aquaporin Abcam Cat#ab78486 Goat polyclonal anti-Podoplanin R&D Cat#AF3244 AF647 donkey anti-rabbit IgG Jackson ImmunoResearch Labs Cat#711-605-152 AF488 donkey anti-rabbit IgG Jackson ImmunoResearch Labs Cat#711-545-152 AF647 donkey anti-goat IgG Jackson ImmunoResearch Labs Cat#705-605-147 AF488 donkey anti-goat IgG Jackson ImmunoResearch Labs Cat#705-545-147 Deposited data RNA Seq raw data This paper GEO Software Image J 1.51 s NIH https://imagej.nih.gov/ij/ Graphpad Prism 8.0 Graphpad Software https://www.graphpad.com/scientificsoftware/prism/ Photoshop Adobe https://www.adobe.com/products/photoshop.html Recombinant DNA ApoM-Fc fusion construct ( Swendeman et al., 2017 ) PMID: 28811382 Chemicals, Peptides, and Recombinant Proteins Tamoxifen Sigma-Aldrich Cat#T5648 Corn Oil Sigma-Aldrich Cat#C8267 Collagenase I Roche Cat#11088793001 Dispase II Roche Cat#04942078001 RIPA Lysis Buffer Santa Cruz Cat#sc-364162 Phosphatase Inhibitor Cocktail Roche Cat#04906837001 Protease Inhibitor Cocktail Roche Cat#04693159001 Lipofectamine™ RNAiMAX Transfection Reagent Invitrogen Cat#13778150 EndoGRO-VEGF Complete Culture Media Kit Millipore Cat#SCME002 DMEM Gibco Cat#11965092 DAPI Invitrogen Cat#D1306 Ketamine Akorn NDC:59399-114-10 Xylazine Akorn NDC:59399-110-20 PFA Electron Microscopy Sciences Cat# 50-980-487 Hydrochloric acid Fisher Scientific Cat# A481-212 Tween80 Sigma-Aldrich Cat#P1754 ApoM-Fc ( Swendeman et al., 2017 ) PMID: 28811382 Critical Commercial Assays Mouse ApoM Elisa Kit Mlbio Biotechnology Cat# {"type":"entrez-nucleotide","attrs":{"text":"ML722506","term_id":"1767879886","term_text":"ML722506"}} ML722506 Hydroxyproline (Hyp) Kit Abcam Cat# ab222941 RNeasy Mini Kit QIAGEN Cat# 74104 EDU Proliferation Kit BD Pharmingen Cat# 565456 Oligonucleotides shRNA targeting sequence: Gankyrin 5’- CCGGG CAGCT TCGAA GAATA GGCAT CTCGA GATGC CTATT CTTCG AAGCT GCTTT TTG -3’ This paper N/A Open in a separate window Key Resources Table

Techniques: Transgenic Assay, Knock-Out, RNA Sequencing Assay, Software, Recombinant, Construct, Lysis, Protease Inhibitor, Transfection, Electron Microscopy, Enzyme-linked Immunosorbent Assay, shRNA, Sequencing

Journal: PLOS Pathogens

Article Title: Endoplasmic reticulum-anchored nonstructural proteins drive human astrovirus replication organelle formation

doi: 10.1371/journal.ppat.1013538

Figure Lengend Snippet: (A) Representative immunofluorescence staining of rHAstV1-V5 infected (MOI = 3; 24 hpi) Cos7 cells expressing the mCherry-KDEL ER marker. Percent maximum intensity profiles of dsRNA (yellow), mCherry-KDEL (magenta), and V5 (cyan) staining were obtained across a 40 µm linescan shown in merged images (white line) using FIJI ImageJ software. (B) Quantification of the colocalization of V5-tagged nsps with remodeled ER and (C) dsRNA. Analysis was performed by calculating the Pearson correlation coefficient (PCC) for n = 15 cells from each rHAstV1-V5 group and (D) pooling of all virus-infected groups (total n = 45) to quantify colocalization of dsRNA with remodeled ER across 3 independent experiments.

Article Snippet: Cos7 cells (a gift from Dr. Alexa Mattheyses, University of Alabama at Birmingham) and HEK293T cells (ATCC, CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 100 IU penicillin per 100 μg/mL streptomycin.

Techniques: Immunofluorescence, Staining, Infection, Expressing, Marker, Software, Virus

Journal: PLOS Pathogens

Article Title: Endoplasmic reticulum-anchored nonstructural proteins drive human astrovirus replication organelle formation

doi: 10.1371/journal.ppat.1013538

Figure Lengend Snippet: (A) Schematic of nsp1a/1 and nsp1a/2 membrane topology and construction of dual-tagged HAstV1 expression plasmids. Nsp1a/1 (light gray), nsp1a/2 (dark gray). Black arrowheads represent sites cleaved by host signal peptidase (SPase). Numbers indicate HAstV1 amino acid positions. (B) Representative immunofluorescence staining of Cos7 cells transfected with the indicated constructs or infected with rHAstV1-V5-1a/1 (MOI = 3) at 24 hours post treatment. Scale bars represent 20 µm. (C) Quantification of the total area of ER signal and (D) mean area of ER particles between groups by particle analysis using FIJI ImageJ software and one-way ANOVA with multiple comparisons versus mock treated cells (*** p < 0.0001). Thresholding was applied uniformly between groups for n = 20 cells across 3 independent experiments. Calnexin was used as an ER marker. (E) Transmission electron microscopy (TEM) of sectioned Cos7 cells transfected with the indicated constructs and fixed at 24 hours post transfection. Yellow arrowheads highlight typical ER membrane morphology. ER whorl (W), convoluted membranes (CM), high curvature ER (HCER). Yellow asterisks (*) indicate representative vesicles with a double membrane component. Scale bars represent 500 µm.

Article Snippet: Cos7 cells (a gift from Dr. Alexa Mattheyses, University of Alabama at Birmingham) and HEK293T cells (ATCC, CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 100 IU penicillin per 100 μg/mL streptomycin.

Techniques: Membrane, Expressing, Immunofluorescence, Staining, Transfection, Construct, Infection, Particle Size Analysis, Software, Marker, Transmission Assay, Electron Microscopy

Journal: PLOS Pathogens

Article Title: Endoplasmic reticulum-anchored nonstructural proteins drive human astrovirus replication organelle formation

doi: 10.1371/journal.ppat.1013538

Figure Lengend Snippet: (A) Construction of dual-tagged HAstV-VA1 expression plasmids. Black arrowheads represent sites cleaved by host signal peptidase (SPase). Numbers indicate VA1 amino acid positions. (B) Representative immunofluorescence staining of Cos7 cells transfected with the indicated constructs at 24 hours post transfection. Scale bars represent 20 µm. Arrowheads indicate regions of over- (yellow) or undersaturation (white) during imaging of nsp1a/1. (C) Quantification of the total area of ER signal and (D) mean area of ER particles between groups by particle analysis using FIJI ImageJ software and one-way ANOVA with multiple comparisons versus GFP-transfected cells (*** p < 0.0001). Thresholding was applied uniformly between groups for n = 20 cells across 3 independent experiments. Calnexin was used as an ER marker.

Article Snippet: Cos7 cells (a gift from Dr. Alexa Mattheyses, University of Alabama at Birmingham) and HEK293T cells (ATCC, CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 100 IU penicillin per 100 μg/mL streptomycin.

Techniques: Expressing, Immunofluorescence, Staining, Transfection, Construct, Imaging, Particle Size Analysis, Software, Marker

Journal: Cell host & microbe

Article Title: Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis.

doi: 10.1016/j.chom.2017.11.009

Figure Lengend Snippet: Figure 2. Rapid PMN Intravascular Chemotaxis to Sequestered C. albicans Is Complement Dependent (A) Lung C5a levels were determined using ELISA in control and C3/ animals, following i.v. C. albicans. Two-way ANOVA, Sidak’s multiple comparison. (B) Using intravital microscopy, PMN chemotactic behaviors in C3/ and anti-C5aR mAb-treated mice were observed. Images shown are 10 min after C. albicans injection. Red arrows highlight yeast not recognized by PMN. Scale bar represents 30 mm. (C) Quantification of PMN chemotaxing to pathogens in C3/ and C5a receptor-blocked mice. One-way ANOVA, Dunnett’s multiple comparison. (D) The percentage of C. albicans phagocytosed during the initial 10 min of intravenous administration in control, C3/, and anti-C5aR mAb-treated mice using pulmonary intravital microscopy. t tests were used to compare C57BL/6 and anti-C5aR mAb treated (*) or C3/ (#) mouse time points. (E and F) C. albicans CFU was quantified in organs at (E) 1 hr and (F) 24 hr after injection with 1 3 106 C. albicans in mice treated with anti-C5a receptor antibodies or isotype. (G) Clinical sepsis scores of isotype and anti-C5aR mAb antibody treated mice 24 hr after injection with 1 3 106 C. albicans. A score of 21 is the clinical endpoint for euthanization. For (A)–(F), at least three individual experiments were performed; for (G) three individual experiments were performed. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Ly6G (clone 1A8) BioLegend RRID: AB_2563207 (BioLegend Cat. No. 127636) Anti-CD31 (clone MEC13.3) BioLegend RRID: AB_2161030 (BioLegend Cat. No. 102515) Anti-CD45 (clone 30-F11) BioLegend RRID: AB_493532 (BioLegend Cat. No. 103121) Anti-CD49b (clone HMa2) BD Bioscience Catalog No. 558295 Anti-C5aR (CD88, clone 20/70) BioLegend RRID: AB_2067286 (BioLegend Cat. No. 135804) Purified anti-mouse Ly6G (clone 1A8) BioXCell BP0075-1 Anti-TER119 Biolegend RRID: AB_528961 (BioLegend Cat. No. 116218) Anti-human CD15 (clone W6D3) BioLegend RRID: AB_756018 (BioLegend Cat. No. 323012) Anti-human CD45 (clone 2D1) R&D Systems Catalog # FAB1430G Anti-human C5aR (CD88, clone S5/1) BioLegend RRID: AB_2259318 (BioLegend Cat. No. 344302) Bacterial and Virus Strains C. albicans (strain ATCC58716) ATCC LUMC-101 Chemicals, Peptides, and Recombinant Proteins Zymosan A (S. cerevisiae) BioParticles ThermoFisher Z23373 Escherichia coli (K-12 strain) BioParticles ThermoFisher E13231 SYTO 9 Green Fluorescent Nucleic Acid Stain ThermoFisher S34854 Compstatin Tocris Cat. No. 2585 LY293111 Cayman Chemical Company CAS N 161172-51-6 Recombinant mouse C5a R&D Systems 2150-C5-025 Critical Commercial Assays LTB4 Parameter Assay Kit R&D Systems KGE006B EasySep Mouse Biotin Positive Selection Kit STEMCELL Technologies Catalog # 18556 Mouse Complement C5a PicoKine ELISA Kit Boster Biological Technology EK0987 Polydimethylsiloxane (PDMS) microfluidic devices SynvivoBio Cat # 104001 Experimental Models: Cell Lines human lung microvascular endothelial cells HLMEC Lonza CC-2527 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Stock No: 000664 LTB4R knockout (B6.129S4-Ltb4r1tm1Adl/J) The Jackson Laboratory Stock No: 008102 C3 / ; (B6;129S4-C3tm1Crr/J) Gift from Paul Kubes N/A CD11b / ; (B6.129S4-Itgamtm1Myd/J) Gift from Paul Kubes N/A CD11a / ; (B6.129S7-Itgaltm1Bll/J) Gift from Paul Kubes N/A LysM-GFP Gift from Paul Kubes N/A Ly6G-cre (C57BL/6-Ly6g(tm2621(CretdTomato)Arte)) Gift from Matthias Gunzer N/A Software and Algorithms Volocity PerkinElmer N/A Leica LAS Leica N/A Prism 7 software GraphPad V7.02 FlowJo FlowJo V10.1 e1 Cell Host & Microbe 23, 1–13.e1–e4, January 10, 2018

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Control, Comparison, Intravital Microscopy, Injection

Journal: Cell host & microbe

Article Title: Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis.

doi: 10.1016/j.chom.2017.11.009

Figure Lengend Snippet: Figure 4. PMN Vascular Clustering during Candidemia Is Dependent on LTB4 In Vivo (A) PMN clustering (white) is compared among C57BL/6, C3/, and anti-C5aR mAb-treated mice following 1 hr of i.v. live C. albicans. Scale bar, 200 mm. (B and C) (B) Mean cluster area and (C) max skeletal length of clusters were compared. One-way ANOVA, Holm-Sidak’s multiple comparison. (D and E) Lung LTB4 levels of C57BL/6 and C3/ mice (D), two-way ANOVA, Holm-Sidak’s multiple comparison, and following anti-C5aR mAb-treated mice (E). (F) LTB4 in isolated pulmonary PMN with or without C. albicans infection (1 hr). Unpaired two-tailed t test. (G) A representative image of PMN clusters (white) in LTB4R/ mice following C. albicans (1 hr). Scale bar, 200 mm. (H and I) (H) Mean cluster area and (I) max. skeletal length of PMN clusters after C. albicans (1 hr). (J–L) 1 3 107 A488 zymosan or 1 3 107 A488 E. coli bioparticles (green) were injected i.v. during lung imaging. (J) Intravital images are shown at 60 min. Scale bar, 100 mm. Clusters were quantified as (K) max skeletal length and (L) mean cluster area. ANOVA was performed for panels with more than two groups, while unpaired two-tailed t test were used to compare two groups. n = at least 3 for each panel. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Ly6G (clone 1A8) BioLegend RRID: AB_2563207 (BioLegend Cat. No. 127636) Anti-CD31 (clone MEC13.3) BioLegend RRID: AB_2161030 (BioLegend Cat. No. 102515) Anti-CD45 (clone 30-F11) BioLegend RRID: AB_493532 (BioLegend Cat. No. 103121) Anti-CD49b (clone HMa2) BD Bioscience Catalog No. 558295 Anti-C5aR (CD88, clone 20/70) BioLegend RRID: AB_2067286 (BioLegend Cat. No. 135804) Purified anti-mouse Ly6G (clone 1A8) BioXCell BP0075-1 Anti-TER119 Biolegend RRID: AB_528961 (BioLegend Cat. No. 116218) Anti-human CD15 (clone W6D3) BioLegend RRID: AB_756018 (BioLegend Cat. No. 323012) Anti-human CD45 (clone 2D1) R&D Systems Catalog # FAB1430G Anti-human C5aR (CD88, clone S5/1) BioLegend RRID: AB_2259318 (BioLegend Cat. No. 344302) Bacterial and Virus Strains C. albicans (strain ATCC58716) ATCC LUMC-101 Chemicals, Peptides, and Recombinant Proteins Zymosan A (S. cerevisiae) BioParticles ThermoFisher Z23373 Escherichia coli (K-12 strain) BioParticles ThermoFisher E13231 SYTO 9 Green Fluorescent Nucleic Acid Stain ThermoFisher S34854 Compstatin Tocris Cat. No. 2585 LY293111 Cayman Chemical Company CAS N 161172-51-6 Recombinant mouse C5a R&D Systems 2150-C5-025 Critical Commercial Assays LTB4 Parameter Assay Kit R&D Systems KGE006B EasySep Mouse Biotin Positive Selection Kit STEMCELL Technologies Catalog # 18556 Mouse Complement C5a PicoKine ELISA Kit Boster Biological Technology EK0987 Polydimethylsiloxane (PDMS) microfluidic devices SynvivoBio Cat # 104001 Experimental Models: Cell Lines human lung microvascular endothelial cells HLMEC Lonza CC-2527 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Stock No: 000664 LTB4R knockout (B6.129S4-Ltb4r1tm1Adl/J) The Jackson Laboratory Stock No: 008102 C3 / ; (B6;129S4-C3tm1Crr/J) Gift from Paul Kubes N/A CD11b / ; (B6.129S4-Itgamtm1Myd/J) Gift from Paul Kubes N/A CD11a / ; (B6.129S7-Itgaltm1Bll/J) Gift from Paul Kubes N/A LysM-GFP Gift from Paul Kubes N/A Ly6G-cre (C57BL/6-Ly6g(tm2621(CretdTomato)Arte)) Gift from Matthias Gunzer N/A Software and Algorithms Volocity PerkinElmer N/A Leica LAS Leica N/A Prism 7 software GraphPad V7.02 FlowJo FlowJo V10.1 e1 Cell Host & Microbe 23, 1–13.e1–e4, January 10, 2018

Techniques: In Vivo, Comparison, Isolation, Infection, Two Tailed Test, Injection, Imaging

Journal: Cell host & microbe

Article Title: Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis.

doi: 10.1016/j.chom.2017.11.009

Figure Lengend Snippet: Figure 7. Anti-fungal Activity and Clustering of Human PMN Is Complement and LTB4 Dependent (A) Images of C5aR inhibition on PMN and C. albicans interactions in a human lung microvascular chamber (anti-C5aR-antibody 20 mg/mL). (B) C. albicans phagocytosis by PMN. (C) Residual immobilized but non-phagocytosed yeast PMN. (D and E) (D) Cluster formation and (E) the number of clustered PMN. (F) Images of LTB4R inhibition on PMN and C. albicans interactions in a human lung microvascular chamber. (G) Quantification of immobilized but non-phagocytosed yeast at 30 min. (H and I) (H) Clusters per field of view (FOV) and (I) PMN that were clustered per FOV were determined at 30 min. All data are expressed as mean ± SEM for n = 5–7. Student’s t tests were used to compare data between two groups. *p < 0.05 and **p < 0.01. All scale bars represent 20 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Ly6G (clone 1A8) BioLegend RRID: AB_2563207 (BioLegend Cat. No. 127636) Anti-CD31 (clone MEC13.3) BioLegend RRID: AB_2161030 (BioLegend Cat. No. 102515) Anti-CD45 (clone 30-F11) BioLegend RRID: AB_493532 (BioLegend Cat. No. 103121) Anti-CD49b (clone HMa2) BD Bioscience Catalog No. 558295 Anti-C5aR (CD88, clone 20/70) BioLegend RRID: AB_2067286 (BioLegend Cat. No. 135804) Purified anti-mouse Ly6G (clone 1A8) BioXCell BP0075-1 Anti-TER119 Biolegend RRID: AB_528961 (BioLegend Cat. No. 116218) Anti-human CD15 (clone W6D3) BioLegend RRID: AB_756018 (BioLegend Cat. No. 323012) Anti-human CD45 (clone 2D1) R&D Systems Catalog # FAB1430G Anti-human C5aR (CD88, clone S5/1) BioLegend RRID: AB_2259318 (BioLegend Cat. No. 344302) Bacterial and Virus Strains C. albicans (strain ATCC58716) ATCC LUMC-101 Chemicals, Peptides, and Recombinant Proteins Zymosan A (S. cerevisiae) BioParticles ThermoFisher Z23373 Escherichia coli (K-12 strain) BioParticles ThermoFisher E13231 SYTO 9 Green Fluorescent Nucleic Acid Stain ThermoFisher S34854 Compstatin Tocris Cat. No. 2585 LY293111 Cayman Chemical Company CAS N 161172-51-6 Recombinant mouse C5a R&D Systems 2150-C5-025 Critical Commercial Assays LTB4 Parameter Assay Kit R&D Systems KGE006B EasySep Mouse Biotin Positive Selection Kit STEMCELL Technologies Catalog # 18556 Mouse Complement C5a PicoKine ELISA Kit Boster Biological Technology EK0987 Polydimethylsiloxane (PDMS) microfluidic devices SynvivoBio Cat # 104001 Experimental Models: Cell Lines human lung microvascular endothelial cells HLMEC Lonza CC-2527 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Stock No: 000664 LTB4R knockout (B6.129S4-Ltb4r1tm1Adl/J) The Jackson Laboratory Stock No: 008102 C3 / ; (B6;129S4-C3tm1Crr/J) Gift from Paul Kubes N/A CD11b / ; (B6.129S4-Itgamtm1Myd/J) Gift from Paul Kubes N/A CD11a / ; (B6.129S7-Itgaltm1Bll/J) Gift from Paul Kubes N/A LysM-GFP Gift from Paul Kubes N/A Ly6G-cre (C57BL/6-Ly6g(tm2621(CretdTomato)Arte)) Gift from Matthias Gunzer N/A Software and Algorithms Volocity PerkinElmer N/A Leica LAS Leica N/A Prism 7 software GraphPad V7.02 FlowJo FlowJo V10.1 e1 Cell Host & Microbe 23, 1–13.e1–e4, January 10, 2018

Techniques: Activity Assay, Inhibition

Journal: Nature

Article Title: Specification of claustro-amygdalar and palaeocortical neurons and circuits.

doi: 10.1038/s41586-024-08361-5

Figure Lengend Snippet: Fig. 1 | Regulation of VLp development and Tfap2d expression by SOX4 and SOX11. a, Coronal sections from VLp of mouse brains from wild-type (WT), Sox4-cKO, Sox11-cKO and Sox4/Sox11-cdKO mice. Nissl staining highlights the BLC. In situ hybridization for Lmo3, Mef2c and Etv1 marks BLC, LA and BLA, whereas GFP and SATB1 immunostaining labels BLC. White and black filled arrowheads highlight normal phenotypes and open arrowheads highlight defective phenotypes. Scale bar, 200 μm. b, Intersection of differentially expressed genes in Sox4-cKO, Sox11-cKO and Sox4/Sox11-cdKO cortex and amygdala compared with controls (n = 3 per genotype). c, Top 50 genes with decreased expression in Sox4/Sox11-cdKO samples over Sox4-cKO, Sox11-cKO and control samples. FC, fold change. d, In situ hybridization for Tfap2d in the VLp of control, Sox4-cKO, Sox11-cKO and Sox4/Sox11-cdKO brains. Scale bars, 500 μm. e, Quantitative PCR showing the percentage of input of six potential Tfap2d enhancers (E1–E6) after ChIP on PCD15.5 wild-type and Sox11-cKO cortices,

Article Snippet: GFP (1:500; Abcam, ab13970; Chicken polyclonal IgY, IHC, Kaur etal, PMID: 32707082) RFP (1:500, Abcam, ab124754; Kaur etal, PMID: 32707082) anti-cleaved capase3 (1:500; Cell Signaling, Cat. 9611, rabbit polyclonal IgG, IHC, Shim et al. 2012 (PMID: 22678282)) anti-SOX11 (Abcam, ab229185, 1080158-1), PolII (Sigma-Aldrich, 05-623),IgG (Diagenode, C15410206) ADGRE1 (1:500, Biorad, MCA497RT, Shi et al., PMID: 31197172), Alexa Fluor® 594 AffiniPure Donkey Anti-Goat IgG,1:250- 1:500,Jackson ImmunoResearch, Catalog No. 705-585-147, Lot No.169491 Alexa Fluor® 594 AffiniPure Donkey Anti-Rabbit IgG, 1:250- 1:500, Jackson ImmunoResearch, Catalog No.711-585-152, Lot No.171656 Alexa Fluor® 647 AffiniPure Donkey Anti-Mouse IgG, 1:250- 1:500, Jackson ImmunoResearch, Catalog No.715-605-151, Lot No.168259 Alexa Fluor® 594 AffiniPure Donkey Anti-Mouse IgG, 1:250- 1:500, Jackson ImmunoResearch, Catalog No.715-585-150, Lot No.167435 Alexa Fluor® 488 AffiniPure Donkey Anti-Chicken IgY, 1:250- 1:500, Jackson ImmunoResearch, Catalog No.703-545-155, Lot No.168728 Alexa Fluor® 594 AffiniPure Donkey Anti-Goat IgG (H+L), 1:250- 1:500, Jackson ImmunoResearch, Catalog No. 705-585-003 Alexa Fluor® 647 AffiniPureTM Donkey Anti-Goat IgG (H+L), 1:250- 1:500, Jackson ImmunoResearch, Catalog No.705-605-003 Donkey anti mouse - 594, 1:250- 1:500, Jackson ImmunoResearch, Catalog No.715-585-150 Alexa Fluor® 594 AffiniPureTM Goat Anti-Rabbit IgG (H+L), 1:250- 1:500, Jackson Immunoresearch, Catalog No.111-585-003 Alexa Fluor® 647 AffiniPureTM Donkey Anti-Goat IgG (H+L), 1:250- 1:500, Jackson ImmunoResearch, Catalog No.705-605-003, Lot No.705605003 Validation SOX11 antibody was validated using the SOX11 KO mice ( Figure 1e, Extended data figure 2) Eukaryotic cell lines Policy information about cell lines and Sex and Gender in Research Cell line source(s) Neuro2a mouse neuroblastoma cell line was purchased from ATCC.

Techniques: Expressing, Staining, In Situ Hybridization, Immunostaining, Control, Real-time Polymerase Chain Reaction

Journal: The Review of scientific instruments

Article Title: Atom Probe Tomography Using an Extreme Ultraviolet Trigger Pulse

doi: 10.1063/5.0160797

Figure Lengend Snippet: (a) Schematic of the EUV beamline. The EUV light is generated collinearly with the NIR driving field in a noble gas filled capillary. The EUV light is isolated from the NIR light by means of two flat mirrors (M1 and M2) and Al filters (i.e. foils). When a toroid (T1) is inserted into the beam path, the light is reflected from a grating (G1) and focused onto a CCD camera for spectral analysis. When a toroid (T1) is removed from the beam path the EUV light transmits past the toroid and through a circular aperture (A1) before reflecting off a flat mirror (M3) and then a toroid (T2) which focuses the light inside the APT analysis chamber and onto the APT specimen apex. During alignments utilizing NIR light, the ultra high vacuum region is separated from the beamline via a sapphire window mounted on a gate valve (GV1). During experiments with EUV light, the ultra high vacuum analysis region is separated from the beamline via an Al filter mounted on a gate valve (GV2). When the instrument is idle, a standard steel gate valve (GV3) is closed to provide robust isolation between the ultrahigh vacuum chamber and the beamline. Two EUV sensitive photodiodes can be inserted into the beampath (PD1, PD2) for power measurements. The focal spot is steered onto the tip by a computer controlled tip/tilt actuator on which a toroid (T2) is mounted. (b) The beamline setup with each of the major components identified.

Article Snippet: The imaging spectrometer consists of the toroid (T1), grating (G1) set up for conical diffraction (500/mm and plane-ruled at 4.7°), and CCD camera (Greateyes, ALEX-s 2k512 BI) set up to image the exit of the capillary to the CCD camera in a 1:1 imaging configuration.

Techniques: Generated, Isolation

Journal: The Review of scientific instruments

Article Title: Atom Probe Tomography Using an Extreme Ultraviolet Trigger Pulse

doi: 10.1063/5.0160797

Figure Lengend Snippet: (a) Calculated reflectivity (p-pol, 6° grazing angle-of-incidence) of the coatings used on the grazing incidence optics present in the beamline. Note that nominally Si coated optics are expected to have a native oxide after exposure to air around 1 nm thick. (b) Calculated transmission (normal angle of incidence) for potentially oxidized Al filters present in the beamline. The dashed lines are calculated for 200 nm and 1 μm thick unoxidized Al. The solid lines are for the same filters with 16 nm and 33 nm of Al2O3 added, respectively. The hollow circles and squares show the measured transmission of two Al filters present in the beamline that are nominally 200 nm and 1 μm thick, respectively. (c) Calculated beamline throughput for various configurations including at the CCD camera and at the APT specimen location. Note that these calculations are smoothed by the spacing of the EUV harmonics (3 eV). (d) The spectra ‘as collected’ on the CCD for EUV light generated with different noble gases with two 1 μm Al filters (Xe) and three 1 μm filters (Kr and Ar). (e) The ‘at source’ spectra calculated by dividing the ‘as collected’ spectra by the beamline throughput to the CCD. (f) The ‘at APT specimen’ spectra calculated by multiplying the ‘at source’ spectra by the beamline throughput to the APT chamber (with a 1 μm Al filter inserted).

Article Snippet: The imaging spectrometer consists of the toroid (T1), grating (G1) set up for conical diffraction (500/mm and plane-ruled at 4.7°), and CCD camera (Greateyes, ALEX-s 2k512 BI) set up to image the exit of the capillary to the CCD camera in a 1:1 imaging configuration.

Techniques: Transmission Assay, Generated

Journal: The Review of scientific instruments

Article Title: Atom Probe Tomography Using an Extreme Ultraviolet Trigger Pulse

doi: 10.1063/5.0160797

Figure Lengend Snippet: (a) Schematic of a 2D beam profile measurement. The EUV light strikes a Ce:YAG crystal and the fluorescence is imaged onto a CCD camera at a ≈ 45° viewing angle. (b) An example 2D beam profile after the image is corrected for viewing angle. Here the beam size is comparable to the resolution of the camera imaging optics making quantitative analysis difficult. (c) Schematic of a ‘knife-edge’ beam profile measurement. Here a grounded local electrode puck was modified to have a flat metal surface intersect the beam. A piece of razor blade is mounted on a specimen puck held at +2.5 kV and slowly translated through the beam as the photoemission current is measured. The resulting one-dimensional profiles (d-e) are fit to a Gaussian beam model to extract the fwhm parameters.

Article Snippet: The imaging spectrometer consists of the toroid (T1), grating (G1) set up for conical diffraction (500/mm and plane-ruled at 4.7°), and CCD camera (Greateyes, ALEX-s 2k512 BI) set up to image the exit of the capillary to the CCD camera in a 1:1 imaging configuration.

Techniques: Fluorescence, Imaging, Modification