For immunofluorescence, embryos were fixed in 4% paraformaldehyde/PBS overnight at 4°C. Embryos were labeled by immunofluorescence as described previously (Boskovski et al., 2013 (link)). All embryos were mounted in Pro-Long Gold (Invitrogen) before imaging. Imaging was performed on a Zeiss Axiovert microscope equipped with Apotome optical interference imaging to obtain optical sections. We employed the following antibodies: anti-acetylated tubulin (Sigma, no. T-6793) (Piperno and Fuller, 1985 (link)) or mouse monoclonal anti-acetylated tubulin, clone 6-11B-1 (1:1,000) (Boskovski et al., 2013 (link)). For secondary antibody we use goat anti-mouse IgG with Alexa Flour 488 conjugate (Life technologies, Item # A-11001, 1:500 dilution).
Alexa flour 488 conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 conjugate is a fluorescent labeling reagent manufactured by Thermo Fisher Scientific. It is used to covalently attach the Alexa Fluor 488 dye to proteins or other biomolecules for detection and visualization purposes in various biological applications.
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Lab products found in correlation
Axiovert microscope, by Zeiss (2 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Fv1000 flowview confocal microscope, by Olympus (1 mentions)
Fish skin gelatin, by Merck Group (1 mentions)
1 2 diamino 4 5 methylenedioxybenzene, by Merck Group (1 mentions)
Chlorpromazine hydrochloride, by Solarbio (1 mentions)
Amiloride hydrochloride, by Solarbio (1 mentions)
Anti β actin, by Abmart (1 mentions)
Brefeldin a, by Selleck Chemicals (1 mentions)
Mtt detection kit, by Solarbio (1 mentions)
Bafilomycin a1, by LC Laboratories (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Bay 11 7082, by Beyotime (1 mentions)
4 protocols using alexa flour 488 conjugate
1
Immunofluorescence Labeling of Embryos
2
Fluorescent Imaging of U87vIII Cells
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U87vIII cells were fixed with 4 % Paraformaldehyde, lysed with 0.2 % Triton X-100 buffer, blocked with 1 % BSA, then incubated in primary antibodies (anti-FLNA, anti-VCL, anti-RICTOR, anti-mTOR, FITC-phalloidin) overnight at 4 °C or 3 h at room temperature. Anti-mouse (Texas Red conjugate), and anti-rabbit (Alexa Flour 594 conjugate and Alexa Flour 488 conjugate) secondary antibodies from Life Technology were used. Nuclei of cells were stained by DAPI. FLNA, Actin filaments, RICTOR, and VCL, were observed using a fluorescence microscope.
3
Immunofluorescence Imaging of Mayaro Virus in HeLa Cells
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HeLa cells seeded onto glass coverslips were infected with MAYV at a multiplicity of infection (MOI) of 1. At 24 h after infection, cells were fixed in 2% paraformaldehyde for 20 min, and permeabilized with 0.25% Triton-X100. Cells were then incubated in 2% bovine serum albumin solution in PBS for 20 min and stained overnight at 4 °C with a mouse immune ascitic fluid anti-MAYV antibody (kindly provided by Dr. Scott Weaver, WRCEVA, UTMB, USA). Finally, cells were washed with PBS buffer and incubated with a Goat anti-mouse secondary antibody, Alexa-Flour 488 conjugate (Invitrogen, Carlsbad, CA, USA), for one hour in the dark. Coverslips were mounted on slides with Prolong Diamond Antifade Mountant with Dapi (Invitrogen, Carlsbad, CA, USA), and the images were obtained with a FV1000 Flowview Confocal microscope (Olympus, Lombard, IL, USA). The pictures were exported and analyzed with ImageJ software [47 (link)]. The number of MAYV-positive cells were counted in at least 10 fields and represented as the percentage of positive cells.
4
Investigating Neu5Gc Uptake and Trafficking in Mammalian Cells
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Neu5Gc was purchased from Aladdin (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum, penicillin, streptomycin, phosphate-buffered saline (PBS), Hank’s Balanced Salt Solution (HBSS) were purchased from BI (Kibbutz Beit-Haemek, Israel). Mouse anti-ZO-1 antibody Alexa Flour 488 conjugate were purchased from Invitrogen (San Francisco, CA, USA). Anti-ZO-1 and occludin antibodies were purchased from Affinity (Changzhou, China). Anti-claudin-1, p65, p-p65, I-κBα, p-I-κBα, and anti-β-actin antibodies were purchased form Abmart (Shanghai, China). BAY 11-7082 was purchased from Beyotime (Shanghai, China). Fish skin gelatin, 1,2-diamino-4,5-methylene-dioxybenzene (DMB), and sodium azide was purchased from Sigma (St. Louis, MI, USA). Chlorpromazine hydrochloride, amiloride hydrochloride and the MTT Detection Kit were purchased from Solarbio (Beijing, China). Dynasore was purchased from MCE (Monmouth Junction, NJ, USA). Nocodazole and nystatin were purchased from Yuanye Bio-Tech (Shanghai, China). Monensin sodium salt and cytochalasin D were purchased from Aladdin (Shanghai, China). Bafilomycin A1 was purchased from LC Laboratories (Woburn, MA, USA). Brefeldin A was purchased from Selleckchem (Houston, TX, USA).
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