For the analysis of virus-induced CPE, plasma membranes of infected cells were stained with Wheat Germ Agglutinin Alexa Fluor 488 Conjugate (WGA, Thermo Fisher Scientific, 1:250) for 20 min, followed by fixation with 4% PFA for 20 min, permeabilization with 0.05% Triton X-100 in PBS for 15 min, and nuclei counter-staining with Hoechst (Thermo Fisher Scientific) for 15 min. Cells were washed twice with PBS and imaged using EVOS fluorescence microscope. For the analysis of CP localization, the infected Huh7.5.1 cells were incubated for 24 h, fixed and permeabilized as above. CP was detected using anti-CPNTD MLB1 antibody followed by incubation with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit, Thermo Fisher, A21441). Nuclei were counter-stained with Hoechst. The images are single plane images taken with a Leica SP5 Confocal Microscope using a water-immersion 63× objective.
Wheat germ agglutinin alexa fluor 488 conjugate wga
Manufactured by Thermo Fisher Scientific
Wheat Germ Agglutinin Alexa Fluor 488 Conjugate (WGA) is a fluorescently labeled lectin that binds to N-acetylglucosamine and sialic acid residues on cell surfaces. It is commonly used as a marker for glycosylated proteins and cell membrane components.
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2 protocols using wheat germ agglutinin alexa fluor 488 conjugate wga
1
Analysis of Virus-Induced Cellular Changes
2
Cardiac Histology and Ultrastructure Analysis
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Mice were euthanized via CO2 asphyxiation, perfused with HBSS (Thermo), followed by heart dissection. Hearts were further incubated for 5 min in 60 mM KCl for synchronization of heart cycle before fixation. Tissues were fixed in 4% PFA at 4 °C overnight and further processed for paraffin sectioning following standard dehydration and embedding protocols. Adult thyroids were sectioned at 14 μm thickness, adult hearts at 10 μm thickness. Masson's Trichrome staining was performed by the Monash Histology Facility. For myocyte area measurements, transverse sections were stained with Wheat Germ Agglutinin Alexafluor 488 conjugate (WGA, Thermo). All images were obtained using Olympus DotSlide (Japan). Mitochondrial density was analyzed by immunofluorescence on 20 μm heart cryosections using TOMM20 rabbit polyclonal antibody (Abcam) raised against the Translocase of Outer Protein Membrane 20 gene (Tomm20). Confocal Z-stack images were performed using Leica SP8. Myocyte area and fluorescent intensity were measured using FIJI software with three replicates for each genotype. For TEM, 50 mg of cardiac tissue was processed by the JAX histology core, and sections were evaluated using JM-1230 microscope (JEOL) coupled to AMT 2K camera (Advanced Microscopy Techniques). Statistical significance of data was determined by ANOVA or Student's t-test.
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