rabbit anti-rat igg Search Results


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  • 99
    Vector Laboratories biotinylated anti rabbit antibody
    Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by <t>biotinylated</t> (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p
    Biotinylated Anti Rabbit Antibody, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 2141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher anti rabbit igg
    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. <t>Biotinylated</t> anti-rabbit <t>IgG</t> followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.
    Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 7812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals rabbit polyclonal antibody
    Association with hWDR79 is required for CB localization of a human scaRNA. ( A ) Subcellular localization of hWDR79 in HeLa cells using rabbit <t>polyclonal</t> antibody. ( B ) Mislocalization of a CAB box mutant ACA scaRNA. Either WT or L1+2mt ACA57 (see
    Rabbit Polyclonal Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 950 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp conjugated anti rabbit igg
    A. Western blot analysis of adult S . japonicum worm extracts obtained on day 6 following treatment with SjIR dsRNAs. Results are shown for proteins recognised by anti-Sm-Pmy antibody (top panel), anti-SjLD1 (middle panel) and anti-SjLD2 (bottom panel) antibodies. The intensity of Sm-Pmy expression was evaluated so as to determine equal protein loading. The arrows indicate the diminished level of SjIR proteins relative to the luciferase treatment control in the first lane. The experiment was repeated twice with similar results obtained. B . Western blot analysis showing no immunological cross reactivity between recombinant HIR and the SjLDs. Commercial recombinant human insulin receptor (rHIR) and recombinant SjLD1 (rSjLD1) and SjLD2 (rSjLD2) were electrophoresed on SDS-PAGE gels, blotted to membrane and probed with rabbit anti-HIR polyclonal antibody (left panel), rabbit anti-SjLD1 (middle panel) and anti-SjLD2 (right panel) as primary antibodies with anti-rabbit <t>IgG</t> conjugated to horseradish peroxidise used as secondary antibody.
    Hrp Conjugated Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti rabbit igg antibodies
    Co-localization of SUMO-1 with NZFP in the nuclear speckles. COS-7 cells over-expressing Myc-SUMO-1 and GFP-NZFP or triple mutant of NZFP fused with GFP were fixed and subjected to immunofluorescence staining with anti-Myc monoclonal antibody. The red signal (SUMO-1) was produced by using <t>Cy3-conjugated</t> anti-mouse <t>IgG,</t> whereas the green signal (wild type or mutant form of NZFP) was visualized by GFP fluorescence.
    Anti Rabbit Igg Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher peroxidase conjugated anti rabbit igg
    <t>Bas1p</t> contains a small domain (BIRD) with interaction and regulatory functions. ( A ) Expression level of the deletion mutants of Bas1p in the bas1bas2 yeast strain. The bas1bas2 (L4233) strain was transformed with the empty control plasmid (vector) or YPL-BAS1 plasmids carrying the full-length (FL) or the deletion version of the BAS1 gene. Total yeast protein extracts were prepared as described in Materials and Methods. Proteins were subjected to SDS–PAGE and electroblotted on a polyvinylidene difluoride membrane. The blot was then incubated with anti-Bas1p purified antibodies (1 µg <t>IgG/ml)</t> followed by horseradish peroxidase-labeled IgG (1/6000 dilution) as secondary antibodies and, finally, with luminescent substrate before exposure to film. The cross-reacting bands are marked by asterisks. Comparable levels of the different Bas1p proteins were also observed in a gcn4bas1BAS2 strain (not shown). ( B ) In vivo effects of the deletions in Bas1p assayed with the ADE1 – LacZ reporter. The bas1BAS2 (Y329, yellow and red boxes) and bas1bas2 (L4233, light and dark blue boxes) cells were co-transformed with a plasmid carrying the ADE1 – LacZ fusion and the different YPL-BAS1 plasmids carrying the full-length (FL) or deleted BAS1 gene. The lane ‘Effector –’ corresponds to transformation of yeast strains with the centromeric vector not carrying the BAS1 gene (YPL). Transformed cells were grown in the presence (red and dark blue boxes) or absence (yellow or light blue boxes) of adenine and β-Gal assays were performed as described in Materials and Methods. ( C ) In vivo role of BIRD in the Bas1p–Bas2p interaction monitored by a two-hybrid approach. The Y187 yeast strain containing an ADE2 -expressing plasmid (pAZ11) (27) was transformed with a centromeric bait plasmid expressing Gal4p DBD fusions (pDBT) (16) and with a second prey plasmid expressing (yellow and red boxes) or not (light and dark blue boxes) the Bas2p–VP16 chimera. Cells were grown in the presence (red and dark blue boxes) or absence (yellow and light blue boxes) of adenine in SC medium lacking histidine and tryptophan. β-Gal assays were performed as described in Materials and Methods.
    Peroxidase Conjugated Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories anti rabbit
    <t>Bas1p</t> contains a small domain (BIRD) with interaction and regulatory functions. ( A ) Expression level of the deletion mutants of Bas1p in the bas1bas2 yeast strain. The bas1bas2 (L4233) strain was transformed with the empty control plasmid (vector) or YPL-BAS1 plasmids carrying the full-length (FL) or the deletion version of the BAS1 gene. Total yeast protein extracts were prepared as described in Materials and Methods. Proteins were subjected to SDS–PAGE and electroblotted on a polyvinylidene difluoride membrane. The blot was then incubated with anti-Bas1p purified antibodies (1 µg <t>IgG/ml)</t> followed by horseradish peroxidase-labeled IgG (1/6000 dilution) as secondary antibodies and, finally, with luminescent substrate before exposure to film. The cross-reacting bands are marked by asterisks. Comparable levels of the different Bas1p proteins were also observed in a gcn4bas1BAS2 strain (not shown). ( B ) In vivo effects of the deletions in Bas1p assayed with the ADE1 – LacZ reporter. The bas1BAS2 (Y329, yellow and red boxes) and bas1bas2 (L4233, light and dark blue boxes) cells were co-transformed with a plasmid carrying the ADE1 – LacZ fusion and the different YPL-BAS1 plasmids carrying the full-length (FL) or deleted BAS1 gene. The lane ‘Effector –’ corresponds to transformation of yeast strains with the centromeric vector not carrying the BAS1 gene (YPL). Transformed cells were grown in the presence (red and dark blue boxes) or absence (yellow or light blue boxes) of adenine and β-Gal assays were performed as described in Materials and Methods. ( C ) In vivo role of BIRD in the Bas1p–Bas2p interaction monitored by a two-hybrid approach. The Y187 yeast strain containing an ADE2 -expressing plasmid (pAZ11) (27) was transformed with a centromeric bait plasmid expressing Gal4p DBD fusions (pDBT) (16) and with a second prey plasmid expressing (yellow and red boxes) or not (light and dark blue boxes) the Bas2p–VP16 chimera. Cells were grown in the presence (red and dark blue boxes) or absence (yellow and light blue boxes) of adenine in SC medium lacking histidine and tryptophan. β-Gal assays were performed as described in Materials and Methods.
    Anti Rabbit, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 2197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno fitc conjugated anti rabbit igg
    Collapse of the nucleocytoplasmic Ran gradient causes an equilibration of RanBP1 across the nuclear envelope. The nuclei of BHK-21 cells were microinjected with <t>FITC-dextran</t> (0.2 mg/ml) as a site-of-injection marker (left) along with either wild-type (WT) Ran (1.0 mg/ml; a panels) or RanGAP (0.4 mg/ml; b panels) and incubated for 10 min at 37°C. The intracellular distribution of endogenous RanBP1 was assessed by indirect immunofluorescence (IF) with anti-RanBP1 and Texas red-conjugated to anti-goat <t>IgG</t> (right) as described in Materials and Methods. Arrows are drawn to help distinguish injected cells from uninjected cells.
    Fitc Conjugated Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories rabbit anti rat igg
    HE staining and <t>B220</t> and <t>IgG</t> deposit detection on testicular sections from control ( a–d ) and TGC-induced EAO ( e–h ) mice. Testes histological sections from control ( a , b ) and TGC-induced EAO ( e , f ) mice were stained with hematoxylin and eosin. Additional tissue sections were incubated with specific antibodies to detect B220 ( c , g ) and IgG deposits ( d , h ) in testes from control ( c , d ) and EAO ( g , h ) mice. The presence of infiltrating B-cells ( g ) and IgG deposits ( h ) with disrupted spermatogenesis was observed in the testes from TGC-induced EAO mice. Brown spots indicate positive cells ( g , h ). Scale bar: 150 μm ( a – f ).
    Rabbit Anti Rat Igg, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fitc conjugated anti rabbit igg
    HE staining and <t>B220</t> and <t>IgG</t> deposit detection on testicular sections from control ( a–d ) and TGC-induced EAO ( e–h ) mice. Testes histological sections from control ( a , b ) and TGC-induced EAO ( e , f ) mice were stained with hematoxylin and eosin. Additional tissue sections were incubated with specific antibodies to detect B220 ( c , g ) and IgG deposits ( d , h ) in testes from control ( c , d ) and EAO ( g , h ) mice. The presence of infiltrating B-cells ( g ) and IgG deposits ( h ) with disrupted spermatogenesis was observed in the testes from TGC-induced EAO mice. Brown spots indicate positive cells ( g , h ). Scale bar: 150 μm ( a – f ).
    Fitc Conjugated Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti rabbit
    HE staining and <t>B220</t> and <t>IgG</t> deposit detection on testicular sections from control ( a–d ) and TGC-induced EAO ( e–h ) mice. Testes histological sections from control ( a , b ) and TGC-induced EAO ( e , f ) mice were stained with hematoxylin and eosin. Additional tissue sections were incubated with specific antibodies to detect B220 ( c , g ) and IgG deposits ( d , h ) in testes from control ( c , d ) and EAO ( g , h ) mice. The presence of infiltrating B-cells ( g ) and IgG deposits ( h ) with disrupted spermatogenesis was observed in the testes from TGC-induced EAO mice. Brown spots indicate positive cells ( g , h ). Scale bar: 150 μm ( a – f ).
    Anti Rabbit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti rat igg whole molecule peroxidase antibody
    HE staining and <t>B220</t> and <t>IgG</t> deposit detection on testicular sections from control ( a–d ) and TGC-induced EAO ( e–h ) mice. Testes histological sections from control ( a , b ) and TGC-induced EAO ( e , f ) mice were stained with hematoxylin and eosin. Additional tissue sections were incubated with specific antibodies to detect B220 ( c , g ) and IgG deposits ( d , h ) in testes from control ( c , d ) and EAO ( g , h ) mice. The presence of infiltrating B-cells ( g ) and IgG deposits ( h ) with disrupted spermatogenesis was observed in the testes from TGC-induced EAO mice. Brown spots indicate positive cells ( g , h ). Scale bar: 150 μm ( a – f ).
    Anti Rat Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 557 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rat igg h l cross adsorbed secondary antibody
    HE staining and <t>B220</t> and <t>IgG</t> deposit detection on testicular sections from control ( a–d ) and TGC-induced EAO ( e–h ) mice. Testes histological sections from control ( a , b ) and TGC-induced EAO ( e , f ) mice were stained with hematoxylin and eosin. Additional tissue sections were incubated with specific antibodies to detect B220 ( c , g ) and IgG deposits ( d , h ) in testes from control ( c , d ) and EAO ( g , h ) mice. The presence of infiltrating B-cells ( g ) and IgG deposits ( h ) with disrupted spermatogenesis was observed in the testes from TGC-induced EAO mice. Brown spots indicate positive cells ( g , h ). Scale bar: 150 μm ( a – f ).
    Goat Anti Rat Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3571 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies rabbit anti rat igg
    Immunolocalization of <t>LipY(ΔPE)</t> at the surface of M. bovis BCG. Immunolabelings were performed on whole M. bovis BCG carrying pMV261:: lipY (Δ PE ), deposited on EM Formvar-coated nickel grids prior to labeling (A and B) or onto prefixed bacteria labeled in suspension prior to processing for conventional EM (C to F). In both cases, bacteria were sequentially exposed to rat anti-LipY(ΔPE) antibodies, rabbit anti-rat <t>IgG,</t> and PAO. (A) Whole bacteria on grids exposed to specific antibody, followed by rabbit anti-rat IgG and PAO: the gold particles (300 to 400 per bacterium) are distributed throughout the bacterial surface (arrows). (B) Whole bacteria on grids exposed to rabbit anti-rat IgG and PAO only as a control: bacteria display at most 40 gold particles on their surface (arrow). (C to E) Thin sections of bacteria exposed to specific antibody, followed by rabbit anti-rat IgG and PAO. (C) The outermost layer of the cell wall is labeled (arrow). This is particularly obvious in the enlarged view (D), where the peptidoglycan layer (PG), the thin electron-translucent layer (ETL), and the outermost fibrillar layer (OL) are clearly visible. (E) When the OL has been shed, bacteria are not labeled. (F) Thin sections of bacteria exposed to preimmune rat serum, followed by rabbit anti-rat IgG and PAO: bacteria are not labeled even when the OL is present. Bars in panels A and B = 0.5 μm; Bars in panels C, E, and F = 0.25 μm; Bar in panel D = 0.1 μm.
    Rabbit Anti Rat Igg, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by biotinylated (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p

    Journal: Journal of neuroscience methods

    Article Title: Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo

    doi: 10.1016/j.jneumeth.2009.07.015

    Figure Lengend Snippet: Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by biotinylated (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p

    Article Snippet: Briefly, slices were blocked with 10% NGS containing 0.1% Triton X-100 antibody in PBS for 1 hr and reacted with either anti-Arc (Arg3.1 antiserum, provided by Dietmar Kuhl, dilution 1:1,500) or anti-GFP (Molecular Probes, Cat# , dilution 1:2,000) rabbit antibody in 1% NGS containing PBS overnight at 4 C. After reaction with biotinylated anti-rabbit antibody (Vector laboratory, dilution 1:200), immunoreactions were visualized with the ABC elite kit (Vector laboratory) and DAB development procedure.

    Techniques: Expressing, Mouse Assay, Immunofluorescence, Labeling, Staining

    Both the isotype control and anti-CD49d antibody injections affected brain CD4 immunoreactivity. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. The presence of IgG2b antibody in the brain was assessed by immunostaining using biotinylated anti-mouse IgG2b antibody (8A) . The presence of T cells in the brain was visualized using anti-CD4 antibody and immunoreactivity observed in the striatum (8B) and parietal cortex (8C) was imaged. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets.

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Both the isotype control and anti-CD49d antibody injections affected brain CD4 immunoreactivity. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. The presence of IgG2b antibody in the brain was assessed by immunostaining using biotinylated anti-mouse IgG2b antibody (8A) . The presence of T cells in the brain was visualized using anti-CD4 antibody and immunoreactivity observed in the striatum (8B) and parietal cortex (8C) was imaged. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets.

    Article Snippet: Elite Vectastain ABC avidin and biotin kits, biotinylated anti-rabbit, anti-mouse, and anti-rat antibodies and the Vector VIP kits were from Vector Laboratories Inc. (Burlingame, CA).

    Techniques: Mouse Assay, Injection, Purification, Immunostaining

    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. Biotinylated anti-rabbit IgG followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.

    Journal: The Journal of Neuroscience

    Article Title: Circulating Insulin-Like Growth Factor I Mediates Effects of Exercise on the Brain

    doi: 10.1523/JNEUROSCI.20-08-02926.2000

    Figure Lengend Snippet: Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. Biotinylated anti-rabbit IgG followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.

    Article Snippet: The secondary antibodies that were used were biotinylated goat anti-mouse IgG (1:1000) (Jackson ImmunoResearch, West Grove, PA) or anti-rabbit IgG (1:250–1:1000) (Pierce, Rockford, IL).

    Techniques: Injection, Labeling, Staining, Incubation, Expressing

    Association with hWDR79 is required for CB localization of a human scaRNA. ( A ) Subcellular localization of hWDR79 in HeLa cells using rabbit polyclonal antibody. ( B ) Mislocalization of a CAB box mutant ACA scaRNA. Either WT or L1+2mt ACA57 (see

    Journal: Molecular cell

    Article Title: A Conserved WD40 Protein Binds the Cajal Body Localization Signal of scaRNP Particles

    doi: 10.1016/j.molcel.2009.02.020

    Figure Lengend Snippet: Association with hWDR79 is required for CB localization of a human scaRNA. ( A ) Subcellular localization of hWDR79 in HeLa cells using rabbit polyclonal antibody. ( B ) Mislocalization of a CAB box mutant ACA scaRNA. Either WT or L1+2mt ACA57 (see

    Article Snippet: For immunoprecipitation of Myc- and FLAG-tagged proteins or of human WDR79, monoclonal 9E10 and M2 antibodies (Sigma) or rabbit polyclonal antibody (Novus Biologicals) were used, respectively.

    Techniques: Mutagenesis

    MYPT1 isoforms in pig coronary arteries and resistance arterioles A. MYPT1 E24+/− splice variants (coding for LZ−/LZ+ isoforms) were amplified by conventional PCR using a single pair of primers flanking the alternative exon and separated by gel electrophoresis. Band intensities were directly quantified and the percentage exon-included in each sample was calculated as E24+/total signal. Data is shown as the mean percentage exon included n= 2 each for coronary arteries and arterioles. Bladder and aorta are shown as standards for this assay. B. MYPT1 E24+/− splice variants from purified RNA pools derived from ECs and SMCs from rat mesenteric arteries, n = 2. C. Rabbit polyclonal antibody specific to the MYPT1 LZ− sequence was used to compare MYPT1 LZ− isoforms by Western blot in pig coronary arteries vs. arterioles, n= 2 each. Antibodies against GAPDH and β-actin were used as internal controls.

    Journal: Microvascular research

    Article Title: Myosin phosphatase isoforms and related transcripts in the pig coronary circulation and effects of exercise and chronic occlusion

    doi: 10.1016/j.mvr.2014.02.004

    Figure Lengend Snippet: MYPT1 isoforms in pig coronary arteries and resistance arterioles A. MYPT1 E24+/− splice variants (coding for LZ−/LZ+ isoforms) were amplified by conventional PCR using a single pair of primers flanking the alternative exon and separated by gel electrophoresis. Band intensities were directly quantified and the percentage exon-included in each sample was calculated as E24+/total signal. Data is shown as the mean percentage exon included n= 2 each for coronary arteries and arterioles. Bladder and aorta are shown as standards for this assay. B. MYPT1 E24+/− splice variants from purified RNA pools derived from ECs and SMCs from rat mesenteric arteries, n = 2. C. Rabbit polyclonal antibody specific to the MYPT1 LZ− sequence was used to compare MYPT1 LZ− isoforms by Western blot in pig coronary arteries vs. arterioles, n= 2 each. Antibodies against GAPDH and β-actin were used as internal controls.

    Article Snippet: Primary antibodies included rabbit polyclonal antibodies that specifically recognize the MYPT1 LZ− isoform (1:1000; rabbit polyclonal IgG ( )), β-actin (1:1,000; Novus Biologicals NB600-501), and GAPDH (1:1000; Advanced Immunochemical RGM2).

    Techniques: Amplification, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Purification, Derivative Assay, Sequencing, Western Blot

    A. Western blot analysis of adult S . japonicum worm extracts obtained on day 6 following treatment with SjIR dsRNAs. Results are shown for proteins recognised by anti-Sm-Pmy antibody (top panel), anti-SjLD1 (middle panel) and anti-SjLD2 (bottom panel) antibodies. The intensity of Sm-Pmy expression was evaluated so as to determine equal protein loading. The arrows indicate the diminished level of SjIR proteins relative to the luciferase treatment control in the first lane. The experiment was repeated twice with similar results obtained. B . Western blot analysis showing no immunological cross reactivity between recombinant HIR and the SjLDs. Commercial recombinant human insulin receptor (rHIR) and recombinant SjLD1 (rSjLD1) and SjLD2 (rSjLD2) were electrophoresed on SDS-PAGE gels, blotted to membrane and probed with rabbit anti-HIR polyclonal antibody (left panel), rabbit anti-SjLD1 (middle panel) and anti-SjLD2 (right panel) as primary antibodies with anti-rabbit IgG conjugated to horseradish peroxidise used as secondary antibody.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential

    doi: 10.1371/journal.pntd.0003730

    Figure Lengend Snippet: A. Western blot analysis of adult S . japonicum worm extracts obtained on day 6 following treatment with SjIR dsRNAs. Results are shown for proteins recognised by anti-Sm-Pmy antibody (top panel), anti-SjLD1 (middle panel) and anti-SjLD2 (bottom panel) antibodies. The intensity of Sm-Pmy expression was evaluated so as to determine equal protein loading. The arrows indicate the diminished level of SjIR proteins relative to the luciferase treatment control in the first lane. The experiment was repeated twice with similar results obtained. B . Western blot analysis showing no immunological cross reactivity between recombinant HIR and the SjLDs. Commercial recombinant human insulin receptor (rHIR) and recombinant SjLD1 (rSjLD1) and SjLD2 (rSjLD2) were electrophoresed on SDS-PAGE gels, blotted to membrane and probed with rabbit anti-HIR polyclonal antibody (left panel), rabbit anti-SjLD1 (middle panel) and anti-SjLD2 (right panel) as primary antibodies with anti-rabbit IgG conjugated to horseradish peroxidise used as secondary antibody.

    Article Snippet: Then the membrane was incubated with anti-rabbit IgG-HRP (Invitrogen), diluted 1:3000, as described above.

    Techniques: Western Blot, Expressing, Luciferase, Recombinant, SDS Page

    Co-localization of SUMO-1 with NZFP in the nuclear speckles. COS-7 cells over-expressing Myc-SUMO-1 and GFP-NZFP or triple mutant of NZFP fused with GFP were fixed and subjected to immunofluorescence staining with anti-Myc monoclonal antibody. The red signal (SUMO-1) was produced by using Cy3-conjugated anti-mouse IgG, whereas the green signal (wild type or mutant form of NZFP) was visualized by GFP fluorescence.

    Journal: Molecules and Cells

    Article Title: SUMO Modification of NZFP Mediates Transcriptional Repression through TBP Binding

    doi: 10.1007/s10059-013-2281-1

    Figure Lengend Snippet: Co-localization of SUMO-1 with NZFP in the nuclear speckles. COS-7 cells over-expressing Myc-SUMO-1 and GFP-NZFP or triple mutant of NZFP fused with GFP were fixed and subjected to immunofluorescence staining with anti-Myc monoclonal antibody. The red signal (SUMO-1) was produced by using Cy3-conjugated anti-mouse IgG, whereas the green signal (wild type or mutant form of NZFP) was visualized by GFP fluorescence.

    Article Snippet: Rabbit anti-SUMO-1 (Santa Cruz), mouse anti-Flag (Sigma-Aldrich), mouse anti-GFP (Clontech), mouse anti-HA (Sigma-Aldrich), mouse anti-Myc (Santa Cruz) antibodies, anti-mouse Cy3-conjugated IgG, horseradish peroxidase (HRP)-linked goat anti-mouse and anti-rabbit IgG antibodies (Sigma-Aldrich) were purchased commercially.

    Techniques: Expressing, Mutagenesis, Immunofluorescence, Staining, Produced, Fluorescence

    Immunodetection of recombinant GALNS. prGALNS was detected by western-blot using a polyclonal rabbit anti-GALNS IgG antibody. GALNS was detected in a purified sample of prGALNS (1) and in human leucocytes (2) under non-reduced ( A ) and reduced ( B ) conditions. Molecular weight marker (MW) was running under the same electrophoresis conditions of prGALNS and human leucocytes.

    Journal: Scientific Reports

    Article Title: Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

    doi: 10.1038/srep29329

    Figure Lengend Snippet: Immunodetection of recombinant GALNS. prGALNS was detected by western-blot using a polyclonal rabbit anti-GALNS IgG antibody. GALNS was detected in a purified sample of prGALNS (1) and in human leucocytes (2) under non-reduced ( A ) and reduced ( B ) conditions. Molecular weight marker (MW) was running under the same electrophoresis conditions of prGALNS and human leucocytes.

    Article Snippet: The recombinant GALNS was recognized with a polyclonal rabbit anti-GALNS IgG antibody, produced against a mixture of highly immunogenic human GALNS peptides , followed by incubation with an anti-rabbit IgG coupled with peroxidase (Sigma-Aldrich).

    Techniques: Immunodetection, Recombinant, Western Blot, Purification, Molecular Weight, Marker, Electrophoresis

    Bas1p contains a small domain (BIRD) with interaction and regulatory functions. ( A ) Expression level of the deletion mutants of Bas1p in the bas1bas2 yeast strain. The bas1bas2 (L4233) strain was transformed with the empty control plasmid (vector) or YPL-BAS1 plasmids carrying the full-length (FL) or the deletion version of the BAS1 gene. Total yeast protein extracts were prepared as described in Materials and Methods. Proteins were subjected to SDS–PAGE and electroblotted on a polyvinylidene difluoride membrane. The blot was then incubated with anti-Bas1p purified antibodies (1 µg IgG/ml) followed by horseradish peroxidase-labeled IgG (1/6000 dilution) as secondary antibodies and, finally, with luminescent substrate before exposure to film. The cross-reacting bands are marked by asterisks. Comparable levels of the different Bas1p proteins were also observed in a gcn4bas1BAS2 strain (not shown). ( B ) In vivo effects of the deletions in Bas1p assayed with the ADE1 – LacZ reporter. The bas1BAS2 (Y329, yellow and red boxes) and bas1bas2 (L4233, light and dark blue boxes) cells were co-transformed with a plasmid carrying the ADE1 – LacZ fusion and the different YPL-BAS1 plasmids carrying the full-length (FL) or deleted BAS1 gene. The lane ‘Effector –’ corresponds to transformation of yeast strains with the centromeric vector not carrying the BAS1 gene (YPL). Transformed cells were grown in the presence (red and dark blue boxes) or absence (yellow or light blue boxes) of adenine and β-Gal assays were performed as described in Materials and Methods. ( C ) In vivo role of BIRD in the Bas1p–Bas2p interaction monitored by a two-hybrid approach. The Y187 yeast strain containing an ADE2 -expressing plasmid (pAZ11) (27) was transformed with a centromeric bait plasmid expressing Gal4p DBD fusions (pDBT) (16) and with a second prey plasmid expressing (yellow and red boxes) or not (light and dark blue boxes) the Bas2p–VP16 chimera. Cells were grown in the presence (red and dark blue boxes) or absence (yellow and light blue boxes) of adenine in SC medium lacking histidine and tryptophan. β-Gal assays were performed as described in Materials and Methods.

    Journal: Nucleic Acids Research

    Article Title: Signaling through regulated transcription factor interaction: mapping of a regulatory interaction domain in the Myb-related Bas1p

    doi:

    Figure Lengend Snippet: Bas1p contains a small domain (BIRD) with interaction and regulatory functions. ( A ) Expression level of the deletion mutants of Bas1p in the bas1bas2 yeast strain. The bas1bas2 (L4233) strain was transformed with the empty control plasmid (vector) or YPL-BAS1 plasmids carrying the full-length (FL) or the deletion version of the BAS1 gene. Total yeast protein extracts were prepared as described in Materials and Methods. Proteins were subjected to SDS–PAGE and electroblotted on a polyvinylidene difluoride membrane. The blot was then incubated with anti-Bas1p purified antibodies (1 µg IgG/ml) followed by horseradish peroxidase-labeled IgG (1/6000 dilution) as secondary antibodies and, finally, with luminescent substrate before exposure to film. The cross-reacting bands are marked by asterisks. Comparable levels of the different Bas1p proteins were also observed in a gcn4bas1BAS2 strain (not shown). ( B ) In vivo effects of the deletions in Bas1p assayed with the ADE1 – LacZ reporter. The bas1BAS2 (Y329, yellow and red boxes) and bas1bas2 (L4233, light and dark blue boxes) cells were co-transformed with a plasmid carrying the ADE1 – LacZ fusion and the different YPL-BAS1 plasmids carrying the full-length (FL) or deleted BAS1 gene. The lane ‘Effector –’ corresponds to transformation of yeast strains with the centromeric vector not carrying the BAS1 gene (YPL). Transformed cells were grown in the presence (red and dark blue boxes) or absence (yellow or light blue boxes) of adenine and β-Gal assays were performed as described in Materials and Methods. ( C ) In vivo role of BIRD in the Bas1p–Bas2p interaction monitored by a two-hybrid approach. The Y187 yeast strain containing an ADE2 -expressing plasmid (pAZ11) (27) was transformed with a centromeric bait plasmid expressing Gal4p DBD fusions (pDBT) (16) and with a second prey plasmid expressing (yellow and red boxes) or not (light and dark blue boxes) the Bas2p–VP16 chimera. Cells were grown in the presence (red and dark blue boxes) or absence (yellow and light blue boxes) of adenine in SC medium lacking histidine and tryptophan. β-Gal assays were performed as described in Materials and Methods.

    Article Snippet: Western blot analysis was performed with the ECL Plus™ western blot kit (Amersham) with purified anti-Bas1p (1 µg purified IgG/ml) as primary antibody and peroxidase-conjugated anti-rabbit IgG (diluted 1:6000; Pierce) as secondary antibody.

    Techniques: Expressing, Transformation Assay, Plasmid Preparation, SDS Page, Incubation, Purification, Labeling, In Vivo

    Collapse of the nucleocytoplasmic Ran gradient causes an equilibration of RanBP1 across the nuclear envelope. The nuclei of BHK-21 cells were microinjected with FITC-dextran (0.2 mg/ml) as a site-of-injection marker (left) along with either wild-type (WT) Ran (1.0 mg/ml; a panels) or RanGAP (0.4 mg/ml; b panels) and incubated for 10 min at 37°C. The intracellular distribution of endogenous RanBP1 was assessed by indirect immunofluorescence (IF) with anti-RanBP1 and Texas red-conjugated to anti-goat IgG (right) as described in Materials and Methods. Arrows are drawn to help distinguish injected cells from uninjected cells.

    Journal: Molecular and Cellular Biology

    Article Title: Facilitated Nucleocytoplasmic Shuttling of the Ran Binding Protein RanBP1

    doi:

    Figure Lengend Snippet: Collapse of the nucleocytoplasmic Ran gradient causes an equilibration of RanBP1 across the nuclear envelope. The nuclei of BHK-21 cells were microinjected with FITC-dextran (0.2 mg/ml) as a site-of-injection marker (left) along with either wild-type (WT) Ran (1.0 mg/ml; a panels) or RanGAP (0.4 mg/ml; b panels) and incubated for 10 min at 37°C. The intracellular distribution of endogenous RanBP1 was assessed by indirect immunofluorescence (IF) with anti-RanBP1 and Texas red-conjugated to anti-goat IgG (right) as described in Materials and Methods. Arrows are drawn to help distinguish injected cells from uninjected cells.

    Article Snippet: All other cells were blocked in 3% BSA–PBS; incubated with a 1:500 dilution of anti-GST monoclonal antibody (MAb; Santa Cruz catalog SC138), a 1:200 dilution of anti-Ran or 1:200 dilution of anti-RanBP1 polyclonal antibody (Santa Cruz catalog SC1159) as primary antibody, and Texas red-conjugated anti-mouse immunoglobulin G (IgG), Texas red-conjugated anti-goat IgG, or FITC-conjugated anti-rabbit IgG (all from Jackson ImmunoResearch Laboratories, diluted 1:500), with DAPI (10 ng/ml); and mounted and viewed as described above.

    Techniques: Injection, Marker, Incubation, Immunofluorescence

    HE staining and B220 and IgG deposit detection on testicular sections from control ( a–d ) and TGC-induced EAO ( e–h ) mice. Testes histological sections from control ( a , b ) and TGC-induced EAO ( e , f ) mice were stained with hematoxylin and eosin. Additional tissue sections were incubated with specific antibodies to detect B220 ( c , g ) and IgG deposits ( d , h ) in testes from control ( c , d ) and EAO ( g , h ) mice. The presence of infiltrating B-cells ( g ) and IgG deposits ( h ) with disrupted spermatogenesis was observed in the testes from TGC-induced EAO mice. Brown spots indicate positive cells ( g , h ). Scale bar: 150 μm ( a – f ).

    Journal: Scientific Reports

    Article Title: Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone

    doi: 10.1038/srep35599

    Figure Lengend Snippet: HE staining and B220 and IgG deposit detection on testicular sections from control ( a–d ) and TGC-induced EAO ( e–h ) mice. Testes histological sections from control ( a , b ) and TGC-induced EAO ( e , f ) mice were stained with hematoxylin and eosin. Additional tissue sections were incubated with specific antibodies to detect B220 ( c , g ) and IgG deposits ( d , h ) in testes from control ( c , d ) and EAO ( g , h ) mice. The presence of infiltrating B-cells ( g ) and IgG deposits ( h ) with disrupted spermatogenesis was observed in the testes from TGC-induced EAO mice. Brown spots indicate positive cells ( g , h ). Scale bar: 150 μm ( a – f ).

    Article Snippet: After rinsing in PBS, the sections were incubated with a rat anti-mouse B220 (clone: RA3-6B2, ×200; BD Biosciences) monoclonal antibody, followed by incubation with rabbit anti-rat IgG (Vector Labs, CA, USA) at room temperature.

    Techniques: Staining, Mouse Assay, Incubation

    Immunolocalization of LipY(ΔPE) at the surface of M. bovis BCG. Immunolabelings were performed on whole M. bovis BCG carrying pMV261:: lipY (Δ PE ), deposited on EM Formvar-coated nickel grids prior to labeling (A and B) or onto prefixed bacteria labeled in suspension prior to processing for conventional EM (C to F). In both cases, bacteria were sequentially exposed to rat anti-LipY(ΔPE) antibodies, rabbit anti-rat IgG, and PAO. (A) Whole bacteria on grids exposed to specific antibody, followed by rabbit anti-rat IgG and PAO: the gold particles (300 to 400 per bacterium) are distributed throughout the bacterial surface (arrows). (B) Whole bacteria on grids exposed to rabbit anti-rat IgG and PAO only as a control: bacteria display at most 40 gold particles on their surface (arrow). (C to E) Thin sections of bacteria exposed to specific antibody, followed by rabbit anti-rat IgG and PAO. (C) The outermost layer of the cell wall is labeled (arrow). This is particularly obvious in the enlarged view (D), where the peptidoglycan layer (PG), the thin electron-translucent layer (ETL), and the outermost fibrillar layer (OL) are clearly visible. (E) When the OL has been shed, bacteria are not labeled. (F) Thin sections of bacteria exposed to preimmune rat serum, followed by rabbit anti-rat IgG and PAO: bacteria are not labeled even when the OL is present. Bars in panels A and B = 0.5 μm; Bars in panels C, E, and F = 0.25 μm; Bar in panel D = 0.1 μm.

    Journal: Infection and Immunity

    Article Title: Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY ▿

    doi: 10.1128/IAI.00410-07

    Figure Lengend Snippet: Immunolocalization of LipY(ΔPE) at the surface of M. bovis BCG. Immunolabelings were performed on whole M. bovis BCG carrying pMV261:: lipY (Δ PE ), deposited on EM Formvar-coated nickel grids prior to labeling (A and B) or onto prefixed bacteria labeled in suspension prior to processing for conventional EM (C to F). In both cases, bacteria were sequentially exposed to rat anti-LipY(ΔPE) antibodies, rabbit anti-rat IgG, and PAO. (A) Whole bacteria on grids exposed to specific antibody, followed by rabbit anti-rat IgG and PAO: the gold particles (300 to 400 per bacterium) are distributed throughout the bacterial surface (arrows). (B) Whole bacteria on grids exposed to rabbit anti-rat IgG and PAO only as a control: bacteria display at most 40 gold particles on their surface (arrow). (C to E) Thin sections of bacteria exposed to specific antibody, followed by rabbit anti-rat IgG and PAO. (C) The outermost layer of the cell wall is labeled (arrow). This is particularly obvious in the enlarged view (D), where the peptidoglycan layer (PG), the thin electron-translucent layer (ETL), and the outermost fibrillar layer (OL) are clearly visible. (E) When the OL has been shed, bacteria are not labeled. (F) Thin sections of bacteria exposed to preimmune rat serum, followed by rabbit anti-rat IgG and PAO: bacteria are not labeled even when the OL is present. Bars in panels A and B = 0.5 μm; Bars in panels C, E, and F = 0.25 μm; Bar in panel D = 0.1 μm.

    Article Snippet: Thin sections of even thickness (70 nm) were collected on nickel grids and sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG (Dako) for 1 h, and (iv) protein A coupled to gold particles of 5 nm in diameter (PAO) (University of Utrecht, Utrecht, The Netherlands) for 30 min. Antibodies and PAO were diluted in PBS containing 5% BSA and 0.1% Tween 20.

    Techniques: Labeling

    Localization of LipY and LipY(ΔPE) in M. bovis BCG. (A) Subcellular localization of LipY and LipY(ΔPE) in M. bovis BCG strains. Cultures were lysed and fractionated to separate the cytoplasm (Cy) from the cell wall (CW). Equal amounts of proteins (10 μg) of each fraction were subjected to SDS-PAGE, electroblotted onto a nitrocellulose membrane, and probed with either rat anti-LipY(ΔPE) antiserum (top), monoclonal anti-KatG antibodies (middle), or rabbit anti-OmpATb antiserum (bottom). (B) EM immunolocalization of LipY. Thin sections of cryosubstituted M. bovis BCG LipY(ΔPE) were sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG for 1 h, and (iv) PAO for 30 min. Intracytoplasmic labeling is indicated by arrows and surface labeling by arrowheads. Bar = 0.5 μm.

    Journal: Infection and Immunity

    Article Title: Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY ▿

    doi: 10.1128/IAI.00410-07

    Figure Lengend Snippet: Localization of LipY and LipY(ΔPE) in M. bovis BCG. (A) Subcellular localization of LipY and LipY(ΔPE) in M. bovis BCG strains. Cultures were lysed and fractionated to separate the cytoplasm (Cy) from the cell wall (CW). Equal amounts of proteins (10 μg) of each fraction were subjected to SDS-PAGE, electroblotted onto a nitrocellulose membrane, and probed with either rat anti-LipY(ΔPE) antiserum (top), monoclonal anti-KatG antibodies (middle), or rabbit anti-OmpATb antiserum (bottom). (B) EM immunolocalization of LipY. Thin sections of cryosubstituted M. bovis BCG LipY(ΔPE) were sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG for 1 h, and (iv) PAO for 30 min. Intracytoplasmic labeling is indicated by arrows and surface labeling by arrowheads. Bar = 0.5 μm.

    Article Snippet: Thin sections of even thickness (70 nm) were collected on nickel grids and sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG (Dako) for 1 h, and (iv) protein A coupled to gold particles of 5 nm in diameter (PAO) (University of Utrecht, Utrecht, The Netherlands) for 30 min. Antibodies and PAO were diluted in PBS containing 5% BSA and 0.1% Tween 20.

    Techniques: SDS Page, Incubation, Labeling

    Specific anti-LipY humoral responses in M. tuberculosis -infected patients as opposed to healthy controls. (A) Purification of the recombinant LipY and LipY(ΔPE) proteins expressed in M. smegmatis carrying either pSD26:: lipY or pSD26:: lipY (Δ PE ). Proteins were extracted from M. smegmatis cell wall preparations, excised from preparative polyacrylamide gels, and then electroeluted to obtain pure proteins. (B) ELISA reactivities of IgG and IgM anti-LipY and anti-LipY(ΔPE) antibodies were assayed in sera of either M. tuberculosis -infected group 1 adult patients or healthy controls (HC) ( n = 44 for healthy controls; n = 69 for patients). (C and D) ELISA reactivities of anti-LipY and anti-LipY(ΔPE) antibodies in two different categories of infected children. (C) IgG and IgM reactivities of sera from recently M. tuberculosis -infected children and from healthy controls ( n = 12 for healthy controls; n = 30 for patients). (D) IgG reactivities for patients with extrapulmonary TB ( n = 12 for healthy controls; n = 27 for patients).

    Journal: Infection and Immunity

    Article Title: Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY ▿

    doi: 10.1128/IAI.00410-07

    Figure Lengend Snippet: Specific anti-LipY humoral responses in M. tuberculosis -infected patients as opposed to healthy controls. (A) Purification of the recombinant LipY and LipY(ΔPE) proteins expressed in M. smegmatis carrying either pSD26:: lipY or pSD26:: lipY (Δ PE ). Proteins were extracted from M. smegmatis cell wall preparations, excised from preparative polyacrylamide gels, and then electroeluted to obtain pure proteins. (B) ELISA reactivities of IgG and IgM anti-LipY and anti-LipY(ΔPE) antibodies were assayed in sera of either M. tuberculosis -infected group 1 adult patients or healthy controls (HC) ( n = 44 for healthy controls; n = 69 for patients). (C and D) ELISA reactivities of anti-LipY and anti-LipY(ΔPE) antibodies in two different categories of infected children. (C) IgG and IgM reactivities of sera from recently M. tuberculosis -infected children and from healthy controls ( n = 12 for healthy controls; n = 30 for patients). (D) IgG reactivities for patients with extrapulmonary TB ( n = 12 for healthy controls; n = 27 for patients).

    Article Snippet: Thin sections of even thickness (70 nm) were collected on nickel grids and sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG (Dako) for 1 h, and (iv) protein A coupled to gold particles of 5 nm in diameter (PAO) (University of Utrecht, Utrecht, The Netherlands) for 30 min. Antibodies and PAO were diluted in PBS containing 5% BSA and 0.1% Tween 20.

    Techniques: Infection, Purification, Recombinant, Enzyme-linked Immunosorbent Assay