ty 23  (Bio X Cell)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner

doi: 10.1016/j.jcmgh.2021.01.016

Figure Lengend Snippet: CD73 is expressed primarily on hepatocytes in normal mouse liver. t-distributed stochastic neighbor embedding (tSNE) and violin plots showing RNA sequencing analysis of the mouse liver from ( A and B ) fluorescence-activated cell sorter (FACS)-sorted and ( C and D ) microfluidic droplet captured cells showing the highest expression of the mouse CD73-encoding gene Nt5e in hepatocytes. Data were obtained from Tabula Muris. ( E ) Fresh-frozen liver sections were stained with antibodies against CD73 (green), and the cell-specific markers keratins K8/18 (hepatocyte), K19 (cholangiocyte), CD31 (endothelium), and F4/80 (Kupffer cells) in red. Bottom row : Magnified views of the boxed areas in the top row . Blue, 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei (DNA). ( F ) Immunofluorescence staining for CD73 (green) in frozen liver sections showing co-localization with the bile-canalicular marker zonula occludens 1 (ZO1) (red). Right : 4× magnified view of the boxed area in the merged panel . DAPI-stained nuclei. Blue, DAPI-stained nuclei (DNA). Scale bars : 20 μm. NK, natural killer; LSEC, liver sinusoidal endothelial cell.

Article Snippet: Primary antibodies CD73 (clone TY/23, 550738; BD Pharmingen, San Jose, CA), K19-AF647 (ab192980; Abcam), K8/18 (clone 8592), and zonula occludens 1 (clone D6L1E, 13663; Cell Signaling) in blocking solution were incubated overnight at 4°C.

Techniques: RNA Sequencing Assay, Fluorescence, Expressing, Staining, Immunofluorescence, Marker

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner

doi: 10.1016/j.jcmgh.2021.01.016

Figure Lengend Snippet: Generation of liver-specific CD73-LKO mice. ( A ) PCR analysis of Nt5e generated a 349-bp fragment from the Cre-targeted allele in the CD73-LKO mouse liver. Nt5e (wt/wt) , Nt5e (fl/wt) , and Nt5e (fl/fl) mice are controls. Representative immunoblots of CD73 in ( B ) primary hepatocytes, and ( C ) in total liver lysates from male and female WT and CD73-LKO mice. Actin and tissue nonspecific alkaline phosphatase (TNAP) immunoblots serve as controls. ( D ) Semiquantitative analysis of CD73 protein expression based on immunoblot band intensities in panel C . ∗∗ P < .01, ∗∗∗ P < .001, 2-way analysis of variance. Error bars represent SD. ( E ) Immunofluorescence staining for CD73 (green), tight junction protein zonula occludens 1 (ZO1) (red), and 4′,6-diamidino-2-phenylindole (DAPI)-stained DNA (blue) on frozen liver tissue sections from WT ( top ) and CD73-LKO ( bottom ) mice. Scale bar : 20 μm. ( F ) AMPase activity in WT and CD73-LKO mice using formalin-fixed liver tissue sections. The brown deposits indicate ecto-AMPase activity in the presence of AMP. Stars indicate the central vein. Scale bar : 400 μm.

Article Snippet: Primary antibodies CD73 (clone TY/23, 550738; BD Pharmingen, San Jose, CA), K19-AF647 (ab192980; Abcam), K8/18 (clone 8592), and zonula occludens 1 (clone D6L1E, 13663; Cell Signaling) in blocking solution were incubated overnight at 4°C.

Techniques: Generated, Western Blot, Expressing, Immunofluorescence, Staining, Activity Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner

doi: 10.1016/j.jcmgh.2021.01.016

Figure Lengend Snippet: CD73-LKO mice develop normally and do not show major liver abnormalities up to 21 weeks of age. ( A ) Formalin-fixed liver sections of 7-week-old male mice stained with H&E. Scale bar : 200 μm. ( B ) Comparison of body weight from WT and CD73-LKO mice at 6–21 weeks of age. n = 15–21 mice/group. ( C ) Analysis of liver-to-body weight ratios of 21-week-old mice. Males: n = 18 WT, n = 21 CD73-LKO. Females: n = 9 WT, n = 15 CD73-LKO. Error bars represent SD.

Article Snippet: Primary antibodies CD73 (clone TY/23, 550738; BD Pharmingen, San Jose, CA), K19-AF647 (ab192980; Abcam), K8/18 (clone 8592), and zonula occludens 1 (clone D6L1E, 13663; Cell Signaling) in blocking solution were incubated overnight at 4°C.

Techniques: Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner

doi: 10.1016/j.jcmgh.2021.01.016

Figure Lengend Snippet: CD73-LKO mice develop spontaneous liver injury after 21 weeks of age in a sex-dependent manner. ( A ) Serum analysis of alanine aminotransferase (ALT) in 21- to 22-week-old male (blue) and female (red) WT ( filled circles ) and CD73-LKO mice ( open circles ). Males: n = 8 WT; n = 8 CD73-LKO; P = .08 (unpaired t test); females: n = 7 WT; n = 7 CD73-LKO. P = .3 (unpaired t test). ( B ) Representative H&E images of formalin-fixed liver sections from 22-week-old male and female WT and CD73-LKO mice. Scale bars : 200 μm. ( C ) Representative H&E images of formalin-fixed liver sections from 36- to 42-week-old male WT and CD73-LKO mice. Scale bars : 200 μm. ( D ) Quantification of blinded histologic scoring of hepatocyte degeneration (defined as swelling and ballooning) in 21- to 42-week-old mice. Scoring: 0, none; 4, mild swelling/focal ballooning; 8, severe swelling/extensive ballooning (scores were averaged for animals with ballooning + swelling). ∗ P < .05; 1-way analysis of variance. Box-and-whisker plots of ( E ) albumin and ( F ) blood urea nitrogen (BUN) in male (blue) and female (red) WT ( filled circles ) and CD73-LKO mice ( open circles ). Same animals as in panel D . Males: n = 13 WT; n = 12 CD73-LKO; females: n = 14 WT; n = 18 CD73-LKO mice. ∗ P < .05, ∗∗∗∗ P < .0001; unpaired t test.

Article Snippet: Primary antibodies CD73 (clone TY/23, 550738; BD Pharmingen, San Jose, CA), K19-AF647 (ab192980; Abcam), K8/18 (clone 8592), and zonula occludens 1 (clone D6L1E, 13663; Cell Signaling) in blocking solution were incubated overnight at 4°C.

Techniques: Whisker Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner

doi: 10.1016/j.jcmgh.2021.01.016

Figure Lengend Snippet: Metabolic imbalance and increased steatosis in male CD73-LKO mouse hepatocytes and livers. ( A ) Ingenuity pathway analysis of proteomic results showing activated ( orange ) and inhibited ( blue ) pathways in freshly isolated hepatocytes from male CD73-LKO mice relative to WT controls. ( B ) H&E staining of formalin-fixed liver sections. Magnified view of boxed areas in the left panel comparing the presence of steatosis in male and female CD73-LKO mice. Scale bar : 200 μm. ( C ) Quantification of blinded histologic scoring of microvesicular and macrovesicular steatosis. Scoring: 0, none; 1, mild; 2, moderate; 3, severe (scores were averaged for animals with microsteatosis and macrosteatosis). Males: n = 13 WT; n = 12 CD73-LKO; females: n = 14 WT; n = 18 CD73-LKO. ∗∗∗ P < .001, 1-way analysis of variance. ( D ) Gene expression analysis of lipid metabolism genes Acaca , Hmgcr , Hmgcs1 , Srebp1a , and Srebp1c using total liver messenger RNA in male and female WT and CD73-LKO mice. n = 13 WT male, n = 12 LKO male, n = 14 WT female, n = 18 LKO female. ∗ P < .05, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, 2-way analysis of variance. Error bars represent SD. EIF2, eukaryotic initiation factor 2; NER, nucleotide excision repair; PPARα/RXRα, peroxisome proliferator-activated receptor α/retinoid X receptor α; tRNA, transfer RNA.

Article Snippet: Primary antibodies CD73 (clone TY/23, 550738; BD Pharmingen, San Jose, CA), K19-AF647 (ab192980; Abcam), K8/18 (clone 8592), and zonula occludens 1 (clone D6L1E, 13663; Cell Signaling) in blocking solution were incubated overnight at 4°C.

Techniques: Isolation, Staining, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner

doi: 10.1016/j.jcmgh.2021.01.016

Figure Lengend Snippet: Inhibition of CD73 enzymatic activity promotes lipid accumulation in hepatocytes. ( A ) Isolated WT C57BL/6 male hepatocytes were treated for 24 hours with 1 μmol/L oleic acid in the presence or absence of the CD73 inhibitor, APCP (10 μmol/L). Intracellular lipid accumulation was analyzed using LipidTOX neutral lipid stain ( red ). ( B ) Quantification of lipid accumulation in panel A . ∗∗ P < .01, ∗∗∗∗ P < .0001; 1-way analysis of variance. Error bars represent SD. ( C ) WT mice were fed normal chow (Control) or a HFD for 14 weeks. Top panels : Representative H&E images of Control- and HFD-fed mice, showing the development of steatosis in the HFD condition. Bottom panels : Representative enzyme histochemistry images showing a decrease in CD73 AMPase activity in the HFD condition. Scale bars : 200 μm.

Article Snippet: Primary antibodies CD73 (clone TY/23, 550738; BD Pharmingen, San Jose, CA), K19-AF647 (ab192980; Abcam), K8/18 (clone 8592), and zonula occludens 1 (clone D6L1E, 13663; Cell Signaling) in blocking solution were incubated overnight at 4°C.

Techniques: Inhibition, Activity Assay, Isolation, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner

doi: 10.1016/j.jcmgh.2021.01.016

Figure Lengend Snippet: CD73-generated extracellular adenosine activates AMPK in hepatocytes and livers from CD73-LKO mice and show impaired AMPK signaling. ( A ) Representative immunoblots of AMPK substrate phosphorylation in liver lysates from male and female mice. ( B ) Semiquantitative analysis of AMPK substrate phosphorylation relative to total protein based on immunoblots in panel A . ∗∗∗ P < .0001, 2-way analysis of variance. Error bars represent SD. ( C ) Fresh-frozen liver sections were stained with antibodies against AMPK ( red ) and zonula occludens 1 (ZO1) (green) show similar AMPK distribution in WT and CD73-LKO mice. Bottom panels : Magnified view of the boxed areas . Scale bar : 50 μm. ( D ) Isolated WT hepatocytes were treated with AMP and recombinant soluble CD73 (rCD73) at the indicated concentrations. Representative immunoblots of total and phospho-AMPKα. ( E ) Quantification of phosphorylated/total AMPK. n = 4 replicates. ∗∗ P < .001; 2-way analysis of variance. ( F ) Representative immunoblot analysis showing AMPK phosphorylation in WT hepatocytes treated with AMP, rCD73, and the adenosine transport inhibitor nitrobenzylthioinosine (NBTI). ( G ) Quantification of phosphorylated/total AMPK in rCD73-treated hepatocytes, in the absence/presence of AMP and NBTI. n = 3 replicates. 2-way analysis of variance. Error bars represent SD. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Primary antibodies CD73 (clone TY/23, 550738; BD Pharmingen, San Jose, CA), K19-AF647 (ab192980; Abcam), K8/18 (clone 8592), and zonula occludens 1 (clone D6L1E, 13663; Cell Signaling) in blocking solution were incubated overnight at 4°C.

Techniques: Generated, Western Blot, Staining, Isolation, Recombinant

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner

doi: 10.1016/j.jcmgh.2021.01.016

Figure Lengend Snippet: Loss of hepatocyte CD73 results in spontaneous liver inflammation. ( A ) Representative H&E images of inflammatory lesions in livers from 22-week-old CD73-LKO mice. Boxed areas in the top panels are magnified in the bottom panels . ( B ) Quantification of blinded histologic analysis of portal and lobular inflammation (scores were averaged for animals with portal + lobular inflammation). Scoring: 0, none; 2, minimal; 4, mild; 6, moderate; 8, severe. ( C ) Total liver messenger RNA analysis of proinflammatory markers interleukin 1β and tumor necrosis factor α in male (blue) and female ( red ) mice aged 21–42 weeks. ( D ) Representative images of immunohistochemical-stained paraffin-embedded liver tissues for neutrophil marker Ly6G ( brown ). Harris hematoxylin-stained nuclei (blue). Inset : magnified view of the boxed area showing signal distribution. Scale bar : 200 μm. ( E ) Quantification of Ly6G + cells per view field in panel D . ( F ) Gene expression analysis of ectonucleotidase CD39 ( Entpd1 ) and adenosine receptors A1R, 2AR, 2BR, and 3R ( Adora1 , 2a , 2b , 3 ) using total liver messenger RNA in male and female WT and LKO mice. n = 13 WT male, n = 12 LKO male, n = 14 WT female, n = 18 LKO female. ∗ P < .05, ∗∗ P < .01, ∗∗∗∗ P < .0001, unpaired t test. Error bars represent SD.

Article Snippet: Primary antibodies CD73 (clone TY/23, 550738; BD Pharmingen, San Jose, CA), K19-AF647 (ab192980; Abcam), K8/18 (clone 8592), and zonula occludens 1 (clone D6L1E, 13663; Cell Signaling) in blocking solution were incubated overnight at 4°C.

Techniques: Immunohistochemical staining, Staining, Marker, Expressing

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: (A) Using immunohistochemistry with a CD73-specific antibody and a rhodamin red λ conjugated secondary antibody, confocal microscopy at a magnitude of 10× revealed staining of glomerular cells in the mesangium as well as peritubular cells in renal tissue of WT mice which is absent in the mutant. Images are representative of 3 of 5 animals with more than 2 sections per kidney. (B) Renal function estimated by the measurement of serum creatinine, auto normalized, in WT and CD73 −/− mice. 8 to19 mice per group were used. Data are presented as means ± SEM, level of significance as indicated according to Student t-test. (C) Total proteinuria was elevated and increased with age in CD73 −/− mice as compared to the wild type controls. We detected proteinuria/creatinuria in [g/g], left panel, and total urine protein in [mg/24h], right panel. 8 to 19 mice per group were used. Data are presented as means ± SEM, level of significance as indicated according to Student t-test.

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques: Immunohistochemistry, Confocal Microscopy, Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: (A) Profiling the urinary protein content, 10 µg of total urinary protein from WT and CD73 −/− mice were subjected per lane to a 12.5% nonreducing SDS-PAGE (silver staining). Mice groups are shown as indicated, Alb = bovine serum albumin, M = rainbowmolecule weight marker with characteristic proteins as indicated (CA = carbonic anhydrase, GD = glutamatic dehydrogenase, Myo = myoglobin red). Proteins bands identified by size are indicated: α1-M = α 1-microglobulin, RBP = retinol binding protein. (B) Urinary albumin excretion was not different as compared to WT mice. 9 to 24 mice per group were used. Data are presented as means SEM.

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques: SDS Page, Silver Staining, Marker, Binding Assay

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: Five sections per animal were analyzed using the point-counting method and a 100-point-eyepiece (integration plate II, Zeiss Co.) at a magnitude of 100× (oil immersion). (A) Volume of glomerular capillaries in 10 3 µm 3 in CD73 −/− mice as compared to WT (n = 5−6, means ± SD, p<0.02 according to Student t-test.). (B) Volume of glomerular cells in [µm 3 ] CD73 −/− mice as compared to WT (n = 5−6, means ± SD, p<0.01 according to Student t-test.).

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: (A) The expression of the podocyte markers WT1, synaptopodin and nephrin was immunohistochemically detected on 6 µM cryosections of the renal cortex of CD73 −/− mice vs. WT-mice. Images are representative of 6–7 animals per group. (B) Statistical analysis showed a decreased number of WT1-stained podocytes per glomerulus (counted in 30 glomeruli per section and 7 mice per group), means ± SEM, p<0.0001 according to Student t-test (left panel). Histomorphometrical analysis (integration plate II, Zeiss Co., magnitude of 100×, oil immersion) confirmed reduced podocytes per glomerulus as compared to other glomerular cells (n = 5−6 mice per group), means ± SD, p<0.01 according to Student t-test (right panel).

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: Electrone microscopic analysis was performed n = 7 CD73 −/− mice vs. WT, original magnification x 10000 or 5000. (A) Ultrastructural analysis of the glomerular filtration barrier from a 3 months old WT mouse with regular appearance of foot processes and slit membranes and fenestration of the endothelium. (B) Analysis of the glomerular filtration barrier from a 3 months old CD73 −/− mouse shows cytoplasmic swelling apparent in endothelial cells (cap. Lumen = capillary lumen, ery = erythrocyte), endothelial fenestrations and single foot effacements were reduced (arrow). No subepithelial depositis were detectable. (C) Overview of a glomerular segment with regular appearance and without glomerulitis (3 months old WT-mouse). (D) Overview of a glomerular segment in a 3 months old CD73 −/− mouse (mes = mesangial cell, ly = lymphocytes) shows lymphocytic glomerulitis.

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques: Filtration

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: (A) Representative images of fibrillar collagen deposition shown by Sirius Red staining (in CD73 −/− as compared to WT controlc mice, ×200 magnification). (B) Image analysis demonstrates that CD73 −/− mice exhibit increased levels of fibrillary collagen deposition within the cortex compared with WT controls (significance as indicated, p<0.002 CD73 −/− vs WT mice, Sirius red staining, 3–5 images per animal, n = 7 animals/group, Student t-test).

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques: Staining

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: The renal cortex of CD73 −/− mice showed glomerular deposits of IgG and C3 as well as increased presence of CD 11b- a n d CD8- positive cells as well as CD25- and GR-1–positive cells in the interstitium of CD73 −/− mutants which were absent in WT-mice. (A) IgG staining (green, FITC conjugated secondary antibodies) and C3 staining (red, rhodamin red λ conjugated secondary antibodies) was carried out in cryo-conserved, 7-µm sections of the kidneys of WT and CD73 −/− mice. Images are representative of >3 independent experiments. (B) CD11b and CD 8 staining (green, FITC conjugated secondary antibodies) and CD25 and GR-1 staining (red, Cy 3) was performed in cryo-conserved, 6–7-µm sections of the mice kidneys. Images are representative of 5 to 7 animals with more than 2 sections per kidney.

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques: Staining

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: The levels of IL-18, VEGF, MIP-2 and TNFα were analyzed in serum and kidney tissue of CD73 −/− and WT mice using BioPlex analysis. 7 animals from each mice group were analyzed. Data are presented as mean l ± SEM. Level of significance as indicated according to according to Mann-Whitney-U-test (p<0.005).

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

doi: 10.1371/journal.pone.0037100

Figure Lengend Snippet: Proinflammatory pattern of angiogenetic factors, chemokines and cytokines observed in the serum but not in the renal tissue of CD73 −/− mice.

Article Snippet: Tissue was fixed for 10 min with “Zamboni’s fixative” (4% PFA, 15% picrine acid in phosphate buffer, pH 7.4) and washed three times with PBS before incubation with blocking solution (10% normal goat serum or 10% donkey serum) for 1 h. The following primary antibodies were used: anti-CD73 (clone TY/23; rat, BD Pharmingen), anti-C3 (RMC11H9; rat, monoclonal; Acris Antibodies, Hiddenhausen, Germany), anti-CD8 and -CD11b (goat, polyclonal; Santa Cruz Biotechnology, CA, USA), anti-GR1 (rat anti-mouse Ly-6G and Ly-6C monoclonal, BD Pharmingen), anti-CD 25 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA), anti-WT1 (rabbit anti-mouse, polyclonal, Santa Cruz Biotechnology, CA, USA).

Techniques: