antibody alexa fluor 594 labeled goat anti rabbit igg antibody  (Thermo Fisher)

Journal: PLoS ONE

Article Title: Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis

doi: 10.1371/journal.pone.0163302

Figure Lengend Snippet: Localization of TL and GSLII binding sites in T . cruzi . Endocytosis kinetics of fluorescent Alexa Fluor 594 conjugated Tf was performed in order to follow T . cruzi endocytic pathway from the flagellar pocket/cytostome to the reservosomes. Parasites were fixed at different time points and probed with biotinylated TL (A), biotinylated ricin (B) or Alexa 488 conjugated GSLII (C). The addition of chitin hydrolysate clearly shows inhibition of TL and GSLII staining. (A) Co-localization of biotinylated-TL (green) and Tf (red). (B) Co-localization of biotinylated-ricin (green) and Tf (red). Addition of 200 mM galactose abolished the ricin staining. (C) Co-localization of Alexa 488 conjugated GSLII (green) and Tf (red). (D) Co-localization of Alexa 488 conjugated GSLII (green) and TcJ6 (red). (E) Co-localization of Alexa 488 conjugated GSLII (green) and anti-BiP (red). (F) GSLII blotting of cell extracts enriched by GSLII chromatography. GSLII blots of T . cruzi CHAPS- and Triton-soluble (CHAPS+Triton X-114) cell lysate fractions were enriched by GSLII chromatography and then treated (+) or not (-) with PNGase F. Blots were probed with biotinylated-GSLII. The GSLII blot indicates the presence of N- acetylglucosamine modification in both soluble and membrane fractions. Treatment of the fractions with PNGase F decreased the reactivity of GSLII confirming N -glycoprotein type modification.

Article Snippet: Primary antibodies were detected with an Alexa Fluor 594-conjugated goat anti-rabbit IgG (Life Technologies) washed three times in TBS.

Techniques: Binding Assay, Inhibition, Staining, Chromatography, Modification

Journal: PLoS ONE

Article Title: Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis

doi: 10.1371/journal.pone.0163302

Figure Lengend Snippet: Inhibition of uptake of Tf by TL in epimastigote forms of T . cruzi . Trypanosomes preincubated with biotinylated TL in the presence of 20 μM FMK-024 (25 μg/ml) and in the absence (A, left panel) or presence of competing chitin hydrolysate (A, right panel), were then incubated with Tf Alexa-594 for 5 or 30 min at 27°C. Cells were then fixed and treated for fluorescence microscopy. Similar incubations wherein TL was substituted by GSLII (B) were performed to assess the specificity of the TL labeling. Furthermore, live parasites preincubated with DyLight 488-TL and 20 μM protease inhibitor (FMK-024) for 5 min and then incubated for 60 min in the presence of Alexa Fluor 594 conjugated Tf showed a lectin labeling in the cytostome/cytopharynx (arrowhead), while no Tf labeling (red signal) was observed in these conditions (C, upper panel). In presence of a molar excess of chitin hydrolysate an intense labeling of Tf exclusively concentrate into reservosomes (arrow) while no green signal corresponding to TL was observed anymore (C, lower panel). Inhibition of trypanosomes Tf uptake with TL was furthermore quantified by flow cytometry (D). The TL signal was dropping from 913 to 273 of mfi in the absence or presence of chitin hydrolysate, respectively (D, left histogram). Conversely, Tf signal was increasing from 597 to 3793 of mfi in the absence or presence of chitin hydrolysate, respectively (D, right histogram).

Article Snippet: Primary antibodies were detected with an Alexa Fluor 594-conjugated goat anti-rabbit IgG (Life Technologies) washed three times in TBS.

Techniques: Inhibition, Incubation, Fluorescence, Microscopy, Labeling, Protease Inhibitor, Flow Cytometry, Cytometry

Journal: International Journal of Molecular Sciences

Article Title: The Serine Protease Autotransporters TagB, TagC, and Sha from Extraintestinal Pathogenic Escherichia coli Are Internalized by Human Bladder Epithelial Cells and Cause Actin Cytoskeletal Disruption

doi: 10.3390/ijms21093047

Figure Lengend Snippet: Intracellular localization of TagB, TagC, and Sha determined by confocal microscopy. ( A ) Z-stack imaging showing the localization of TagB, TagC, and Sha and their respective serine active site mutant variants during interaction with 5637 bladder epithelial cells after 5 h of incubation. SPATEs were detected by Alexa Fluor 594 (white arrowheads, red fluorescence) using anti-mouse secondary antibody and actin was stained by Alexa Fluor 488- phalloidin (green fluorescence). Images are displayed in a 3D section view with large Z-sections in the X-Y direction (R), Z-projection in the X–Z direction (P), and Z-projection in the Y–Z direction (Q). The green and red lines in R indicate the orthogonal planes of the X–Z and Y–Z projection. For each selected section, the signal was gathered from a span of 5 μm. Scale bar: 10 µm ( B ) Quantitative analysis of fluorescence intensity of F-actin in the cells treated with native or mutant SPATEs. Analysis of fluorescence intensity was done in green channel by measuring the mean gray value on ImageJ. Data represent the mean ± SEM of at least three independent experiments. Significant differences between fluorescence intensity of each native and mutant SPATE treated cell was determined using Student’s t-test with ** p

Article Snippet: This was followed by incubation with secondary antibody Alexa Fluor 594-labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Confocal Microscopy, Imaging, Mutagenesis, Incubation, Fluorescence, Staining

Journal: PLoS ONE

Article Title: Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

doi: 10.1371/journal.pone.0191052

Figure Lengend Snippet: Characterization of VH411-S-Tag peptide conjugate binding to the CHO-hLDLR-EGFP cell line. (A) Representative confocal photomicrographs of CHO-hLDLR-EGFP and CHO-hTfR-EGFP cells (green) incubated 1 hr at 37°C with 10 μM of VH411-S-Tag detected post-fixation with an anti-S-Tag and A647-conjugated secondary antibody (cyan) and 10 μg/mL of DiI-LDL. Cell nuclei are labeled with Hoechst#33258 (blue) at 0.5 μg/mL. Co-labeling appears in blue-green/orange in the merged pictures. Note that VH411-S-Tag co-localizes with hLDLR and is internalized by CHO-hLDLR-EGFP cells. (B) Representative confocal photomicrographs of CHO-hLDLR-EGFP cells (green) incubated 1 hr at 37°C with a macromolecular complex (see scheme Fig 3B) resulting from the co-incubation and interaction of 10 μM VH411-S-Tag peptide or VH411Sc-S-Tag peptide with the anti-S-Tag antibody (1/200) and the Alexafluor 647-conjugated secondary antibody (1/800) (cyan). Cells were concomitantly exposed to 10 μg/mL of DiI-LDL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in blue-green/orange in the merged pictures.

Article Snippet: Cells were washed with PBS and incubated for 1 hr with primary antibodies directed against the hLDLR (rabbit, 1/100, Acris, Herford, Germany), against M13 bacteriophage (mouse, 1/3000, Sigma-Aldrich), against the S-Tag (goat, 1/250, Abcam) or against the Fc (mouse, 1/100, Jackson ImmunoResearch), followed by 3 washes with PBS and incubation with Alexa fluor 488, 594 or 647 anti-rabbit, anti-mouse or anti-goat secondary antibodies (1/800, Invitrogen).

Techniques: Binding Assay, Incubation, Labeling

Journal: Oncotarget

Article Title: In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors

doi: 10.18632/oncotarget.7062

Figure Lengend Snippet: Inhibition of the nuclear translocation of NF-κB upon GANT-61 treatment Immunofluorescence analysis was performed on MCF-7 and SK-BR-3 breast cancer cells after treatment with GANT-61 (10 μM) or vehicle alone for 24 hours. After the treatment, the cells were fixed, incubated with an anti-NF-κB antibody, washed, and labeled with an Alexa Fluor-488-conjugated goat anti-mouse IgG antibody. The nuclei were counterstained with Hoechst. Original magnification, 400x.

Article Snippet: Goat anti-rabbit IgG Alexa fluor-594-conjugated and goat anti-mouse IgG Alexa fluor-488-conjugated secondary antibody came from Invitrogen (Milano, Italy).

Techniques: Inhibition, Translocation Assay, Immunofluorescence, Incubation, Labeling