Wga af555

Manufactured by Thermo Fisher Scientific

Sourced in United States

The WGA-AF555 is a fluorescent labeling reagent used for the detection and visualization of glycoproteins in various applications, such as Western blotting, immunohistochemistry, and flow cytometry. It binds to N-acetylglucosamine and N-acetylneuraminic acid residues, allowing for the specific labeling of glycosylated proteins.

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Lab products found in correlation

Paraformaldehyde (pfa), by Merck Group (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Lsm 710, by Zeiss (2 mentions) Killik, by Bio-Optica (1 mentions) Scanscope, by Leica Biosystems (1 mentions) Prolong gold antifade solution, by Thermo Fisher Scientific (1 mentions) Formaldehyde, by Polysciences (1 mentions) Lsm 510 meta confocal fluorescence microscope, by Zeiss (1 mentions) Glass slide, by Corning (1 mentions) Ultrapure water, by Merck Group (1 mentions) Alexa fluor 555, by Thermo Fisher Scientific (1 mentions) Sylgard 184 silicone elastomer kit, by Dow (1 mentions) Su 8 2010, by MicroChem (1 mentions) Fd10s, by Merck Group (1 mentions) Wga cf488a, by Biotium (1 mentions)

8 protocols using wga af555

Probiotic Gut Colonization Visualization

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To evaluate probiotic presence in the gut, bacteria were labeled with wheat germ agglutinin-Alexa Fluor 555 conjugate (WGA-AF555) (Thermo Fisher) for 10 min, followed by extensive washes. A dose of 10⁹ CFU bacteria was administered to Lewis rats, and gut samples were excised after 30–60 min, washed with PBS, fixed with paraformaldehyde (PFA, 4% in PBS) for 24 h and then transferred in sucrose (30% in PBS) for cryopreservation. Samples were included in Killik (Bio-Optica) and kept at −80°C, pending analysis. Serial 10 μm thick cryosections were stained with Hematoxilin and Eosin (images digitalized with ScanScope, Aperio technologies) or with the following antibodies: mouse anti-vimentin mAb (V9, Dako), mouse anti-cytokeratin mAb (MNF116, Dako), mouse anti-CD11c mAb (8A2; ThermoFisher), mouse anti-CD3 mAb (G4.18, eBioscience), followed by species-specific Alexa Fluor 488-conjugated secondary antibodies. Isotype control stainings were routinely performed in the immunofluorescence procedures. Nuclei were stained with DAPI (Thermo Fisher Scientific). Single plan and z-scan images were captured via confocal microscopy and Structured Illumination microscopy (SIM), using a 100X APO-TIRF (NA 1.49) objective, with 3D optical sectioning. Images were processed with Fiji software (20 (link)).

Rinaldi E., Consonni A., Cordiglieri C., Sacco G., Crasà C., Fontana A., Morelli L., Elli M., Mantegazza R, & Baggi F. (2019). Therapeutic Effect of Bifidobacterium Administration on Experimental Autoimmune Myasthenia Gravis in Lewis Rats. Frontiers in Immunology, 10, 2949.

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Visualizing RNase J-GFP in bacteria

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Bacterial suspensions of the RNase J-GFP-expressing strain in exponential-growth phase (after 18 h of culture) or in stationary phase (after 40 h of culture) were fixed in 1% paraformaldehyde (Sigma)–PBS for 5 min at room temperature. Following three washes in PBS, bacteria were permeabilized using 0.05% Triton X-100–PBS. Anti-GFP nanobodies coupled with Cy5 (Fluotag-Q; NanoTag Biotechnologies GmbH) were then added (1:250) to the bacterial suspensions and incubated for 1 h at 37°C. The cells were then washed and treated with 2 μg/ml WGA-AF555 (Thermo Fisher Scientific)–PBS. In order to (i) immobilize the bacterial cells onto the coverslips (no. 1.5H; Marienfeld, Germany) and (ii) induce the photoswitching of Cy5, the bacteria were seeded onto STORM buffer-based pads (Blinking Pad kit; Abbelight, Paris, France).

Tejada-Arranz A., Galtier E., El Mortaji L., Turlin E., Ershov D, & De Reuse H. (2020). The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori. mBio, 11(5), e01173-20.

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Visualizing RNase J-GFP in Bacteria

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Bacterial suspensions of the RNase J-GFP expressing strain in exponential growth phase (after 18 hours of culture) or in stationary phase (after 40h of culture), were fixed in 1% paraformaldehyde (Sigma) in PBS for 5 min at room temperature. Following three washes in PBS, bacteria were permeabilized using 0.05% Triton X-100 in PBS. Anti-GFP nanobodies coupled with Cy5 (Fluotag®-Q, NanoTag Biotechnologies GmbH) were then added (1:250) to the bacterial suspensions and incubated for 1h at 37°C. The cells were then washed and treated with 2 µg/mL WGA-AF555 (Thermo Fisher Scientific) in PBS. In order to (i) immobilize the bacterial cells onto the coverslips (#1.5H, Marienfeld, Germany) and (ii) induce the photoswitching of Cy5, the bacteria were seeded onto STORM bufferbased pads (Blinking Pad Kit, Abbelight, Paris, France).

Tejada-Arranz A., Galtier E., Mortaji L.E., Turlin É., Ershov D., & Reuse H.D. (2020). The RNase J-based RNA degradosome is compartmentalized in the gastric pathogenHelicobacter pylori.

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Corneal Neuron Tracing in Mice

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Mice were anesthetized and placed under a stereoscopic dissection microscope. Wheatgerm agglutinin-Alexa Fluor 555 (WGA-AF555, Invitrogen, W32464, USA), a retrograde neural tracer, was used to label TG corneal neurons. Approximately 0.5 μl WGA-AF555 of 5 mg/mL in PBS was injected into the corneal stroma via a glass micropipette (Li et al., 2019 (link)). Five days after WGA-AF555 injection, eDED group mice were held under CDS conditions for 14 days.

Zhang J., Lin H., Li F., Wu K., Yang S, & Zhou S. (2023). Involvement of endoplasmic reticulum stress in trigeminal ganglion corneal neuron injury in dry eye disease. Frontiers in Molecular Neuroscience, 16, 1083850.

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Membrane Staining of Adherent Cells

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Treated cells (50 000) were plated on 24 mm glass coverslips and allowed to attach for 24 h. Cells were then washed with PBS and fixed in 4% formaldehyde (Polysciences, Inc., Warrington, PA) for 30 min. Cells were then washed three times with PBS and membrane-stained with WGA-AF555 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Coverslips were then mounted on microscope slides using Prolong Gold antifade solution (Invitrogen, Carlsbad, CA) containing DAPI for cell nuclei staining. Images were acquired on a LSM 510 Meta confocal fluorescence microscope (Carl Zeiss, Inc., Peabody, MA) with the appropriate filters.

Stephen Z.R., Kievit F.M., Veiseh O., Chiarelli P.A., Fang C., Wang K., Hatzinger S.J., Ellenbogen R.G., Silber J.R, & Zhang M. (2014). Redox-Responsive Magnetic Nanoparticle for Targeted Convection-Enhanced Delivery of O6-Benzylguanine to Brain Tumors. ACS Nano, 8(10), 10383-10395.

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Neutrophil Intracellular Pathogen Visualization

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L. pneumophila-infected neutrophils were incubated for 20 min with WGA-AF555 (Invitrogen) and with Hoechst 33342 (Thermo Scientific) for 5 min for cytoplasmatic membrane and nucleus staining, respectively. Cells were attached to a microscope slide using cytospins and analysed using the Zeiss LSM 710 inverted confocal. The images were obtained using a maximum intensity projection algorithm.

Wee B.A., Alves J., Lindsay D.S., Klatt A.B., Sargison F.A., Cameron R.L., Pickering A., Gorzynski J., Corander J., Marttinen P., Opitz B., Smith A.J, & Fitzgerald J.R. (2021). Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing. Nature Communications, 12, 7165.

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Fluorescent Labeling of Bacterial Cells

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We purchased wheat germ agglutinin lectin labeled with Alexa Fluor 555 (WGA-AF555) or Alexa Fluor 594 (WGA-AF594) from Life Technologies Corp.; Sylgard 184 Silicone Elastomer Kit (poly(dimethylsiloxane) (PDMS)) from Dow Corning Corp.; SU-8 2010 and NanoPG Developer from MicroChem Corp.; Goldseal No. 1 coverslips (25 mm × 50 mm) from Thermo Fisher Scientific, Inc.; and glass slides (50 mm × 50 mm × 1 mm) from Corning, Inc. All other chemicals were purchased from Sigma Aldrich. Complex peptone yeast extract (PYE) medium^{14 (link)} was made from 2 g bactopeptone, 1 g yeast extract, 0.3 g MgSO₄·7H₂O, and 0.0735 g CaCl₂·2H₂O in 1 L of ultrapure water (18 MΩ·cm, Millipore Corp.) and autoclaved before use.

Hoffman M.D., Zucker L.I., Brown P.J., Kysela D.T., Brun Y.V, & Jacobson S.C. (2015). Timescales and Frequencies of Reversible and Irreversible Adhesion Events of Single Bacterial Cells. Analytical chemistry, 87(24), 12032-12039.

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Ventricular Tissue Labeling and Imaging

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For co-labeling with WGA and a volume marker of the extracellular space, we used sections of left ventricular tissue from a control heart and incubated them in modified Tyrode's solution containing 40 μg/ml WGA AF555 (W32464, Life Technologies) for 5-10 min. Subsequently, we placed the tissues in a bath with modified Tyrode's solution and 5mg/ml dextran (molecular weight: 10 kDa) conjugated to fluorescein isothiocyanate (FD10S, Sigma-Aldrich). We used a cotton swab to gently press the endocardial surface of the tissues onto the cover slip at the bottom of the bath.^{23 (link)} The tissues were then imaged through the cover slip using similar approaches as described above.
For evaluation of our approach for vessel reconstruction, a control heart was perfused with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DIL, D-282, Life Technologies) directly after perfusion with modified Tyrode's solution, following an established protocol.^{26 (link)} A tissue section was taken from the right ventricle and labeled with 40 μg/ml WGA CF488A (29022, Biotium) in PBS before compression-free mounting on a cover slip and confocal microscopy.

Seidel T., Edelmann J.C, & Sachse F.B. (2015). Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions. Annals of biomedical engineering, 44(5), 1436-1448.

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