On the day of HIV infection, 2e5 edited CD4+ T cells per stain replicate were collected, fixed in 4% paraformaldehyde for 10 min, and resuspended in 20 μg/mL VVL-FITC (Vector Laboratories #FL-1231-2) and 10 μg/mL GSL-II-AF647 (ThermoFisher #L32451) in 2% FBS in PBS for 1 h at room temperature in the dark. Cells were washed, resuspended in 2% FBS in PBS, and were analyzed by flow cytometry on a BD LSRFortessa X-50 or BD FACSymphony A5.
Vvl fitc
Manufactured by Vector Laboratories
VVL-FITC is a fluorescein isothiocyanate (FITC) conjugate of Vicia villosa lectin. It is a reagent used for the detection and labeling of glycoproteins and cell surface carbohydrates.
Automatically generated - may contain errors
Lab products found in correlation
A1 laser scanning confocal microscope, by Nikon (2 mentions)
M2 medium, by Merck Group (2 mentions)
Ksom hepes buffer, by Merck Group (2 mentions)
Acid tyrode, by Merck Group (2 mentions)
Lsrfortessa x50, by BD (1 mentions)
Facsymphony a5, by BD (1 mentions)
Anti ha, by Thermo Fisher Scientific (1 mentions)
Biotinylated vvl, by Vector Laboratories (1 mentions)
Ha 11, by Fortrea (1 mentions)
Nucleolin 4e2, by Abcam (1 mentions)
Biorevo bz 9000, by Keyence (1 mentions)
6 protocols using vvl fitc
1
Phenotyping HIV-Infected CD4+ T Cells
2
Antibody Localization in Toxoplasma
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The following primary antibodies were used in IFA or Western blot: anti-MIC2 [28 (link)], anti-GRA14 [33 (link)], anti-ROP7 (Monoclonal antibody [MAb] 1B10) [33 (link)], anti-SRS44, anti-IMC3 [34 (link)], mouse anti-ISP3 [10 (link)], anti-IMC1 mAb 45.15 [35 (link)], rat anti-RON11 [24 (link)], VVL-FITC (Vector laboratories), and biotinylated-VVL (Vector laboratories). Hemagglutinin (HA) epitope was detected with mAb HA.11 (Covance) and rabbit polyclonal anti-HA (Invitrogen). For localization of GRASP55, a fluorescent fusion was used as previously described [36 (link)].
3
Immunofluorescence Protocol for Cellular Markers
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Primary antibodies used in this study include rat anti-HA (Roche clone 3F10), VVL-FITC (1:1,000, Vector FL1231); DMC1 antibody (1:10, a kind gift of Christopher Huston, University of Vermont [15 (link)]), rabbit anti-TrpB (31 (link)) and mouse anti-alpha tubulin (a kind gift of Jacek Gaertig, University of Georgia), both used at 1:300; and secondary antibodies carrying a variety of Alexa Fluors (Abcam) at 1:1,000.
4
Blastocyst Isolation and Imaging
5
Blastocyst Isolation and Imaging
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Wild-type 129/SvEv mice were euthanized 3.5 days post-mating. The uterus was isolated and washed in KSOM-HEPES buffer (Milli-pore Sigma). The utero-tubule junctions were cut and the uterine horns were flushed using a 25 gauge needle filled with 0.4 mL of M2 medium (Millipore Sigma). Using a mouth controlled Pasteur pipette, blastocysts were collected and transferred into a drop of PBS. Blastocysts were hatched by serial passage through drops of Acid Tyrodes (Millipore Sigma). Hatched blastocysts were transferred into drops of PBS, and then fixed in 3% paraformaldehyde and VVL immunofluorescence staining was performed as described under Vicia villosa lectin assays. Blastocyst images were obtained using EVOS epifluorescence and Nikon A1 laser scanning confocal microscope. Immunostaining of the trophectoderm of intact, infected blastocysts was performed 48 hours post-infection. The shControl or shGalnt3 infected blastocysts were fixed with 3% paraformaldehyde and blocked for 30 min using 1X carbofree block. Blastocysts were stained for 1 hour using VVL-FITC (Vector Laboratories) at room temperature. The VVL-FITC stained blastocysts were imaged at 20X using EVOS microscopy.
6
Immunofluorescence Staining of Nucleolin
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Cells were cultured on coverslips, fixed with 4 % formaldehyde for 10 min, washed with PBS and afterwards blocked for 1 h in 1 % BSA/PBS-T at RT. The cells were then incubated with the primary antibody (Nucleolin 4E2, Abcam) over night (4 °C) or lectin (VVL-FITC, Vector) for 1 h at RT and washed with PBS. After incubation with the primary antibody, a fluorescent secondary antibody (Alexa Fluor 546 rabbit anti mouse IgG, Life Technologies) was added for 1 h at RT. After washing with PBS, the specimens were observed under a fluorescence microscope (BIOREVO BZ-9000; Keyence) and analyzed with BZ-9000 Generation II Analyzer software.
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