polyclonal rabbit anti human a gal  (Novus Biologicals)

Journal: PLoS ONE

Article Title: Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

doi: 10.1371/journal.pone.0025065

Figure Lengend Snippet: (A) Peroxidase immunohistochemistry for α-Gal A in a biopsy from a male Fabry patient using anti-human α-Gal A antibody. The patient was intravenously infused with α-Gal A 2 h before the biopsy was taken. Labeling of a human glomerulus (G) showing α-Gal A localization in the podocytes (indicated with green arrowheads) and GL-3 inclusions seen as vacuoles (indicated with red arrowheads). Staining is also seen in parietal epithelial cells (indicated with yellow arrows). Scale bar, 25 µm. A high-power view of a portion of the glomerulus (top-right) demonstrates the localization of infused recombinant α-Gal in the podocyte. (B) For comparison, no α-Gal A labeling is seen in the podocytes in a biopsy from an untreated male Fabry patient. (C) The treated male Fabry patient shows no detectable labeling of endogenous α-Gal A in the collecting ducts (CD). Red arrowheads indicate heavy GL-3 inclusions. Scale bar, 25 µm. (D) A normal individual shows labeling of endogenous α-Gal A (green arrowheads) in both thick ascending limbs of Henle (TAL) and in CD. Scale bar, 25 µm. (E) Immunofluorescent demonstration of Alexa-Fluor 488-labeled α-Gal A uptake in human podocytes as a function of time at 37°C. At the indicated times, the cells were fixed and analyzed by confocal microscopy. Scale bar, 10 µm. (F) For colocalization of α-Gal A (green) and lysosomes (red) a merged image is shown. The yellow color illustrates colocalization. Scale bar, 5 µm.

Article Snippet: Recombinant α-Gal A (Fabrazyme) and affinity-purified rabbit polyclonal anti-human α-Gal A were provided by Genzyme Corp. (Framingham, MA, USA).

Techniques: Immunohistochemistry, Labeling, Staining, Recombinant, Confocal Microscopy

Journal: PLoS ONE

Article Title: Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

doi: 10.1371/journal.pone.0025065

Figure Lengend Snippet: (A) Affinity chromatography: the arrowheads indicate the migration of the different α-Gal A binding proteins. (B) Western blot analysis demonstrated that the three bands were megalin, M6PR, and sortilin as indicated with arrows. Lysate from human podocyte culture was used as a positive control.

Article Snippet: Recombinant α-Gal A (Fabrazyme) and affinity-purified rabbit polyclonal anti-human α-Gal A were provided by Genzyme Corp. (Framingham, MA, USA).

Techniques: Affinity Chromatography, Migration, Binding Assay, Western Blot, Positive Control

Journal: PLoS ONE

Article Title: Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

doi: 10.1371/journal.pone.0025065

Figure Lengend Snippet: (A) Human podocytes were incubated with 125 I-α-Gal A for different times. (B) Podocytes incubated with 125 I-α-Gal A for 12 h in the presence or absence of RAP, sortilin propeptide, M6P, and with all three ligands combined at 37°C. Uptake was assayed as described in the method section of the paper. Values are means of triplicate experiments with standard deviations (SD). The addition of sortilin propeptide shows no significant inhibition, however the addition of M6P, RAP or a combination of all three inhibitors show significant reductions (* indicate P<0.001) in the α-Gal A uptake after 12 h.

Article Snippet: Recombinant α-Gal A (Fabrazyme) and affinity-purified rabbit polyclonal anti-human α-Gal A were provided by Genzyme Corp. (Framingham, MA, USA).

Techniques: Incubation, Inhibition

Journal: PLoS ONE

Article Title: Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

doi: 10.1371/journal.pone.0025065

Figure Lengend Snippet: (A) SPR analysis of α-Gal A binding to purified human sortilin and inhibition of binding by NT. The lower binding curve was corrected for the binding of NT itself to sortilin. (B) Uptake of Alexa-Fluor 488-labeled α-Gal A by wild-type and full-length sortilin transfected HEK293 cells in the presence or absence of M6P or M6P and NT for 60 min at 37°C. At the given time, cells were fixed, and analyzed by confocal microscopy. Scale bar, 10 µm.

Article Snippet: Recombinant α-Gal A (Fabrazyme) and affinity-purified rabbit polyclonal anti-human α-Gal A were provided by Genzyme Corp. (Framingham, MA, USA).

Techniques: Binding Assay, Purification, Inhibition, Labeling, Transfection, Confocal Microscopy

Journal: PLoS ONE

Article Title: Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

doi: 10.1371/journal.pone.0025065

Figure Lengend Snippet: (A) Expression of megalin, M6PR, sortilin, podocin, nephrin, APN mRNAs by RT-PCR (+). Negative controls (−) run without RT enzyme. The marker used was 100 bp ladder. (B) Immunoperoxidase labeling of paraffin sections from a normal human kidney incubated with antibodies to megalin, M6PR, and sortilin show that the brush borders of proximal tubules (PT) are heavily stained. The distal tubules (DT) are unstained for megalin, but stained for sortilin and M6PR. The glomeruli (G) show staining for megalin, sortilin and M6PR. Preabsorption of the antibodies with the respective antigens show loss of immunoreactivity, demonstrating antibody specificity. Tubular and podocyte staining was blocked by preincubation. Controls that included only the secondary antibody showed no labeling (data not shown). Scale bar, 50 µm. (C) Dual immunofluorescence and confocal microscopy with monoclonal anti-WT1 antibody (red) and polyclonal antibodies (green) against megalin, sortilin, and M6PR. The respective merge images are shown in both low and high magnifications. Megalin, sortilin, and M6PR are localized in the glomerular podocytes and co-localizes with WT-1 as seen by the merged images indicated with white arrow-heads. High-power views of a portion of a glomerulus show merged images of the receptors and WT1 demonstrating that megalin, sortilin, and M6PR localize primarily to the podocyte cell bodies. Scale bar, 50 µm.

Article Snippet: Recombinant α-Gal A (Fabrazyme) and affinity-purified rabbit polyclonal anti-human α-Gal A were provided by Genzyme Corp. (Framingham, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Labeling, Incubation, Staining, Immunofluorescence, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes—In Vitro Studies

doi: 10.3390/ijms22189683

Figure Lengend Snippet: Immunofluorescence analysis of ex vivo-expanded PEKs stemming from control nontransgenic pigs (CTR nTG). Representative microphotographs depicting the immunofluorescent detection of α1,2-FT-related ( a ) and α-Gal A-related ( b ) locations identified in nontransgenic PEKs. Immunostaining with red Cy3 fluorochrome-tagged or green Alexa Fluor 488 fluorochrome-tagged secondary antibodies and blue fluorescent nuclear counterdyeing with the use of 4′,6-diamidino-2-phenylindole (DAPI). Scale bars represent 100 μm. No positive signaling points descended from either xenogeneic rhα1,2-FT or endogenous species-specific α1,2-FT were recognized ( a ), while sporadic incidence of hardly detectable spots correlated with immunofluorescently labelling the α-Gal A enzymes was noticed ( b ).

Article Snippet: Taking into account the immunoblotting, membranes were blocked for 1 h in 5% non-fat milk in TBST (Tris-buffered saline with 0.1% v / v Tween20; Bioshop Inc.) and, after several rinses with TBST, incubated overnight at +4 °C with the following primary antibodies (the same as those for immunofluorescent labelling): rabbit polyclonal IgGs against human α1,2-FT (diluted 1:1000 in TBST; ab198712, Abcam) and rabbit polyclonal IgGs against human α-Gal A (diluted 1:1000 in TBST; PA5-27349, Thermo Fisher Scientific). β-Actin was used as a reference protein (mouse monoclonal IgG designated as anti-β-actin, diluted 1:2000 in TBST; ab8224, Abcam).

Techniques: Immunofluorescence, Ex Vivo, Immunostaining