Journal:

Article Title: In Vivo Rescue of a Silent tax -Deficient Bovine Leukemia Virus from a Tumor-Derived Ovine B-Cell Line by Recombination with a Retrovirally Transduced Wild-Type tax Gene

doi:

Figure Lengend Snippet: Detection of LTaxSN and BLV sequences in YR2 and YR2LTaxSN cells. (A) Southern blot analysis of SacI-digested genomic DNA. Twenty micrograms of digested DNA prepared from YR2 (lanes 1, 3, 5, and 7) or YR2LTaxSN (lanes 2, 4, 6, and 8) cells was electrophoresed through a 0.8% agarose gel, transferred to nylon membranes, and hybridized with 32P-labelled ENV, NEO, MoMLV LTR, and TAX probes. (B) PCR amplification of LTaxSN and BLV sequences. Genomic DNA isolated from parental YR2 (lanes 1, 5, 9, 13, and 17) and Rat-2 (lanes 4, 8, 12, 16, and 20) cells and from YR2LTaxSN (lanes 2, 6, 10, 14, and 18) and Rat-2LTaxSN (lanes 3, 7, 11, 15, and 19) cells was amplified by PCR with LTaxSN-specific primers P1/T2, P1/P2, T1/P2, and N1/N2 or BLV-specific primers T6/U3 and analyzed by Southern blot hybridization with 32P-labelled TAX and NEO probes.

Article Snippet: FLK (a BLV-infected fetal lamb kidney cell line), Rat-2 (a control cell line, ATCC CRL1764), and Rat-2 derivatives (Rat-2 LTaxSN and Rat-2 NUNL ) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM glutamine, nonessential amino acids, and 100 μg of kanamycin per ml.

Techniques: Southern Blot, Agarose Gel Electrophoresis, Amplification, Isolation, Hybridization

Journal:

Article Title: In Vivo Rescue of a Silent tax -Deficient Bovine Leukemia Virus from a Tumor-Derived Ovine B-Cell Line by Recombination with a Retrovirally Transduced Wild-Type tax Gene

doi:

Figure Lengend Snippet: Detection of BLV and LTaxSN transcripts in YR2LTaxSN cells. (A) Ten micrograms of total RNA isolated from YR2, YR2LTaxSN, Rat-2, Rat-2LTaxSN, or FLK cells was analyzed by Northern blot hybridization with 32P-labelled TAX (left panel) and NEO (right panel) probes. Sizes of mRNAs are indicated. (B) RT-PCR detection of the BLV 2.1-kb doubly spliced transcript in total RNA from YR2 (lanes 1 and 2), YR2NUNL (lanes 3 and 4), Rat-2LTaxSN (lanes 5 and 6), YR2LTaxSN (lanes 7 and 8), and FLK (lanes 9 and 10) cells with two sets of splice-specific primers, EA/C3 (lanes 1, 3, 5, 7, and 9) and EA/T2 (lanes 2, 4, 6, 8, and 10). Amplification products were electrophoresed through a 1% agarose gel, transferred to nylon membranes, and hybridized with a 32P-labelled total BLV probe. Positions and molecular sizes of amplified products are indicated.

Article Snippet: FLK (a BLV-infected fetal lamb kidney cell line), Rat-2 (a control cell line, ATCC CRL1764), and Rat-2 derivatives (Rat-2 LTaxSN and Rat-2 NUNL ) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM glutamine, nonessential amino acids, and 100 μg of kanamycin per ml.

Techniques: Isolation, Northern Blot, Hybridization, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: Immunocytochemical detection of MMCs in epithelial whole mounts and frozen sections of jejunum from T. spiralis-infected BALB/c mice. A to D: Whole mounts of jejunal epithelium were probed with anti-mMCP-1 (pseudo-colored red) or isotype-matched control antibodies together with anti-IgE (pseudo-colored green) or isotype-matched control antibodies and counterstained with DRAQ5 (pseudo-colored blue). IgE surface labeling was almost exclusively restricted to mast cells exhibiting strong intracellular mMCP-1 staining in samples probed with mMCP-1- and IgE-specific antibodies (A and D). Single-negative controls for IgE (B) and mMCP-1 (C) were used to confirm labeling specificity. Frozen sections of jejunum from uninfected (E) and T. spiralis-infected (F) BALB/c mice were probed with rabbit anti-tryptase (pseudo-colored green), anti-IgE (pseudo-colored red), and anti-integrin-α6 antibodies (pseudo-colored blue). No IgE or tryptase-positive cells were detected in uninfected animals (E). Abundant IgE+ve cells were detected in the samples from infected mice, the majority of which were located intraepithelially (F). Although significant numbers of intraepithelial IgE+ve cells were tryptase-negative (F, arrowheads), most IgE+ve cells detected in the lamina propria expressed tryptase (F, arrow). Staining specificity was confirmed using single-positive controls for tryptase (G), integrin-α6 (H), and IgE (I), in which the two other antigen-specific antibodies were replaced with control IgG. Identical excitation and exposure settings were used to acquire images of multilabeled and control samples. Scale bar length (μm) is provided in the bottom left corner of each image.

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Infection, Labeling, Staining

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: IgE+ve cells in the jejunal mucosa of T. spiralis-infected mice express integrin-αEβ7. Frozen sections of jejunum from infected BALB/c mice were triple-labeled with anti-integrin-αE or anti-integrin-β7 together with anti-IgE and anti-integrin-α6 (see Figure 4). Using integrin-α6 staining to differentiate the epithelium (filled bars) and lamina propria (open bars), IgE+ve cells mm−2 (A) and the relative frequencies of IgE+ve cells expressing integrin-αE or integrin-β7 (B) were determined for each location using five randomly selected 0.31-mm2 fields of view per sample. The mean percentage of intraepithelial IgE+ve cells ± 1 SEM is provided in parentheses (A). Data are expressed as mean values ± 1 SEM (n = 4). Differences observed between the epithelial and lamina propria compartments were significant at P < 0.01 (**).

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Infection, Labeling, Staining, Expressing

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: Immunofluorescent detection of integrin-αE and -β7 expression by IgE+ve cells and PCNA-specific staining of toluidine blue-positive mast cells in the jejunal mucosa of S129 β6−/− and β6+/+ mice. A to J: Frozen sections of jejunum were probed with monoclonal rat antibodies specific for integrin-α6 (pseudo-colored blue), IgE (pseudo-colored green), and integrin-β7 or -αE (pseudo-colored red). Representative images are shown from uninfected β6+/+ (A) and β6−/− (B) mice stained for IgE, integrin-α6, and integrin-β7; uninfected β6+/+ (C) and β6−/− (D) mice stained for IgE, integrin-α6, and integrin-αE; T. spiralis-infected β6+/+ (E) and β6−/− (F) mice stained for IgE, integrin-α6, and integrin-β7; and T. spiralis-infected β6+/+ (G) and β6−/− (H) mice stained for IgE, integrin-α6, and integrin-αE. Arrowheads in D and H highlight occasional cells that exhibit low-level integrin-αE immunoreactivity in sections from β6−/− mice (inset in D has been contrast enhanced). Higher magnification images from T. spiralis-infected β6+/+ (I) and β6−/− (J) mice stained for IgE, integrin-α6, and integrin-αE are annotated with arrows (intraepithelial IgE+ve cells) and arrowheads (lamina propria IgE+ve cells). Note that although large numbers of IgE+ve cells are present in the mucosa of T. spiralis-infected β6−/− mice, they are predominantly located in the lamina propria. Single-positive controls from β6+/+ mice for integrin-β7 (K), integrin-αE (L), IgE (M), and integrin-α6 (N), in which two of the three antigen-specific antibodies were substituted with isotype-matched controls, were used to confirm staining specificity. Identical excitation and exposure settings were used to acquire images A to D. Constant excitation and exposure settings were also maintained during acquisition of images E to N. Bright-field images of toluidine blue-labeled mast cells probed with anti-PCNA antibodies from T. spiralis-infected β6+/+ (O) and β6−/− (P) mice, arrow indicates a PCNA-positive mast cell in O. Scale bars, 25 μm.

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Expressing, Staining, Infection, Labeling

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: IgE+ve cell recruitment and integrin-αEβ7 expression in the jejunal mucosa of T. spiralis-infected β6−/− and β6+/+ S129 mice. Frozen sections of jejunum from infected β6−/− (open bars) and β6+/+ (filled bars) S129 mice were triple-labeled with anti-integrin-αE or anti-integrin-β7 together with anti-IgE and anti-integrin-α6 (see Figure 4). Data are presented as mean values ± 1 SEM (n = 3) for IgE+ve cells mm−2 (A); the percentage of IgE+ve cells located intraepithelially (B); and the relative frequencies of IgE+ve cells expressing integrin-αE (C) or integrin-β7 (D). Differences observed between β6−/− and β6+/+ S129 mice were significant at P < 0.05 (*) and P < 0.01 (**).

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Expressing, Infection, Labeling

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: RT-PCR analysis of integrin-αE and -β7 transcript abundance and toluidine blue analysis of MMC distribution in the jejunal mucosa of T. spiralis-infected β6+/+ and β6−/− S129 mice. A: RT-PCR products for integrin-αE, integrin-β7, and the housekeeping gene GAPDH from reverse-transcribed total jejunal RNA from infected β6−/− (lanes 1 to 4) and β6+/+ (lanes 5 to 8) mice. B: RT-PCR product signal intensity expressed as a percentage of GAPDH signal intensity. Differences in PCR product abundance between β6+/+ and β6−/− S129 mice were significant at P < 0.05 (*). Mast cells were identified using toluidine blue staining (pH 0.5) in Carnoy’s-fixed/paraffin-embedded sections from β6+/+ (C) and β6−/− (D) S129 mice. Note that mast cells are present in greater numbers in β6−/− mice but are predominantly restricted to the lamina propria. Scale bars, 50 μm.

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Staining

Journal: Kidney international

Article Title: Anti-DNA auto-antibodies initiate experimental lupus nephritis by binding directly to the glomerular basement membrane in mice

doi: 10.1038/ki.2011.484

Figure Lengend Snippet: Monoclonal Antibody Binding to DNA, Chromatin, and Basement Membrane Antigens.

Article Snippet: Biotinylated goat anti-mouse IgG, IgG 2a , and IgG 2b ; FITC-goat anti-mouse IgG; and FITC-streptavidin were purchased from Southern Biotechnology (Birmingham, AL); alkaline phosphatase-streptavidin, from Jackson Immunoresearch Laboratories (West Grove, PA); biotinylated rat anti-perlecan mAb (clone A7L6), from Lab Vision (Thermo Fisher Scientific, Fremont, CA); Alexa 546-strepatavidin and TO-PRO3 ® DNA dye, from Molecular Probes (Invitrogen, Carlsbad, CA); and anti-C3-FITC, from BD Bioscience.

Techniques: Binding Assay, Membrane, Enzyme-linked Immunosorbent Assay, In Vivo, Activity Assay

Journal: Kidney international

Article Title: Anti-DNA auto-antibodies initiate experimental lupus nephritis by binding directly to the glomerular basement membrane in mice

doi: 10.1038/ki.2011.484

Figure Lengend Snippet: Supernatant mAbs that do not bind BM when assayed alone do not bind BM when combined with supernatant mAbs that do bind BM. Supernatant mAbs from the indicated hybridoma pairs were assayed by direct ELISA for BM binding. Titration curves represent serial dilution of supernatants assayed independently for IgG 2a or IgG 2b binding to BM: solid circles, IgG 2a ; open squares, IgG 2b ; solid lines, IgG 2a and IgG 2b mAbs co-incubated; and broken lines, IgG 2a or IgG 2b mAb incubated alone. Supernatant concentrations of mAbs: 163p.124, 12.1 μg/ml; 163p.132, 34.7 μg/ml; DNA3, 29.1 μg/ml; 163p.77, 23.5 μg/ml; 163p.64, 10.0 μg/ml; 452s.46, 6.4 μg/ml; and 452s.160, 18.7 μg/ml. Maximum OD 405 = 2.600.

Article Snippet: Biotinylated goat anti-mouse IgG, IgG 2a , and IgG 2b ; FITC-goat anti-mouse IgG; and FITC-streptavidin were purchased from Southern Biotechnology (Birmingham, AL); alkaline phosphatase-streptavidin, from Jackson Immunoresearch Laboratories (West Grove, PA); biotinylated rat anti-perlecan mAb (clone A7L6), from Lab Vision (Thermo Fisher Scientific, Fremont, CA); Alexa 546-strepatavidin and TO-PRO3 ® DNA dye, from Molecular Probes (Invitrogen, Carlsbad, CA); and anti-C3-FITC, from BD Bioscience.

Techniques: Direct ELISA, Binding Assay, Titration, Serial Dilution, Incubation

Journal: Kidney international

Article Title: Anti-DNA auto-antibodies initiate experimental lupus nephritis by binding directly to the glomerular basement membrane in mice

doi: 10.1038/ki.2011.484

Figure Lengend Snippet: Detection of glomerular (a) IgG 2b , 163p.77 but not (b) IgG 2a , 452s.160 in kidney serial cryosections 24 hours after co-injecting 1 mg of each purified mAb into a BALB/c mouse. Serial cryosections had granular IgG 2b but no IgG 2a within MM. Mice injected with 163p.64, IgG 2a and 452s.46, IgG 2b (see ) and 163p.124, IgG 2a and 163p.132, IgG 2b had IgG 2a but no IgG 2b staining. Results were similar in replicate mice.

Article Snippet: Biotinylated goat anti-mouse IgG, IgG 2a , and IgG 2b ; FITC-goat anti-mouse IgG; and FITC-streptavidin were purchased from Southern Biotechnology (Birmingham, AL); alkaline phosphatase-streptavidin, from Jackson Immunoresearch Laboratories (West Grove, PA); biotinylated rat anti-perlecan mAb (clone A7L6), from Lab Vision (Thermo Fisher Scientific, Fremont, CA); Alexa 546-strepatavidin and TO-PRO3 ® DNA dye, from Molecular Probes (Invitrogen, Carlsbad, CA); and anti-C3-FITC, from BD Bioscience.

Techniques: Purification, Injection, Staining

Journal: Kidney international

Article Title: Anti-DNA auto-antibodies initiate experimental lupus nephritis by binding directly to the glomerular basement membrane in mice

doi: 10.1038/ki.2011.484

Figure Lengend Snippet: Hybridomas producing BM-binding mAb induce proteinuria.

Article Snippet: Biotinylated goat anti-mouse IgG, IgG 2a , and IgG 2b ; FITC-goat anti-mouse IgG; and FITC-streptavidin were purchased from Southern Biotechnology (Birmingham, AL); alkaline phosphatase-streptavidin, from Jackson Immunoresearch Laboratories (West Grove, PA); biotinylated rat anti-perlecan mAb (clone A7L6), from Lab Vision (Thermo Fisher Scientific, Fremont, CA); Alexa 546-strepatavidin and TO-PRO3 ® DNA dye, from Molecular Probes (Invitrogen, Carlsbad, CA); and anti-C3-FITC, from BD Bioscience.

Techniques: Injection