Cells were treated with PBS or 10 nM Dd-AF647 for 2 h at 37°C. After incubation, cells were fixed with 1× FACS lysing solution (BD Biosciences). Nuclei were stained with DAPI (1:3,000; Sigma) and membranes with Alexa Fluor 555-conjugated wheat germ agglutinin (WGA; 1:200; Invitrogen) during 10 min at room temperature. Images were acquired after sequential laser excitation using an LSM 710 NLO laser-scanning microscope (Zeiss) onto optical sectioning of 1 μm and analyzed with ImageJ software.
Alexa fluor 555 conjugated wheat germ agglutinin wga
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Alexa Fluor 555-conjugated wheat germ agglutinin (WGA) is a fluorescent-labeled lectin that binds to N-acetylglucosamine and sialic acid residues on cell surfaces. It can be used to label and visualize cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti β catenin, by Merck Group (2 mentions)
Anti col1, by Abcam (2 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (2 mentions)
Anti collagen type 2, by Rockland Immunochemicals (2 mentions)
Anti myosin, by Developmental Studies Hybridoma Bank (2 mentions)
Dylight 550 conjugates, by Thermo Fisher Scientific (2 mentions)
Ec plan neofluar 100x 1.30 oil objective, by Zeiss (2 mentions)
Anti fibronectin, by Merck Group (2 mentions)
Axio imager z1, by Zeiss (2 mentions)
Sp5 confocal microscope, by Leica (2 mentions)
Lsm 710 nlo laser scanning microscope, by Zeiss (1 mentions)
Facs lysing solution, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tcs sp8 x confocal microscope, by Leica (1 mentions)
Ab7817, by Abcam (1 mentions)
Dish 35 mm, by Ibidi (1 mentions)
Hc pl apo 20x 0.75 imm, by Leica (1 mentions)
8 protocols using alexa fluor 555 conjugated wheat germ agglutinin wga
1
Visualizing Cell Membrane and Nucleus
2
Confocal Imaging of Fibroblasts on CyPhyGels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For confocal imaging, fibroblasts were cultured on CyPhyGels of different stiffness for 4 days, fixed using 4% paraformaldehyde (Roth, Germany) in PBS for 15 min. Glycoproteins at the cell membrane were stained with AlexaFluor555-conjugated wheat germ agglutinin (WGA, 1 μg/mL, Invitrogen, Germany) in 3% bovine serum albumin in PBS for 1 h, followed by three 10-min washing steps in PBS. Thereafter, cells were permeabilized using 0.5% Triton-X100 (Sigma-Aldrich, Germany) in PBS for 15 min and blocked using 3% bovine serum albumin in PBS for 1 h. Primary antibody against alpha smooth muscle actin (αSMA; ab7817, Abcam, Germany) was applied 1:50 in 1% bovine serum albumin in PBS overnight. After three washing steps in PBS, AlexaFluor488-coupled secondary anti-mouse was applied 1:500 in PBS for 1 h. Nuclear counterstaining was performed using Hoechst-33342 (20 μM, ThermoFisher, Germany) for 2–3 min in PBS. All staining was performed at room temperature.
Imaging was performed on a Leica TCS SP8 X confocal microscope using a 20× multi-immersion objective (HC PL APO 20x/0.75 IMM) with water as immersion medium. For imaging, the CyPhyGels were mounted upside-down in a PBS-filled microscopy dish (µ-Dish 35 mm, ibidi, Germany). Cell area was determined using ImageJ after manually tracing cell borders based on WGA signal.
Imaging was performed on a Leica TCS SP8 X confocal microscope using a 20× multi-immersion objective (HC PL APO 20x/0.75 IMM) with water as immersion medium. For imaging, the CyPhyGels were mounted upside-down in a PBS-filled microscopy dish (µ-Dish 35 mm, ibidi, Germany). Cell area was determined using ImageJ after manually tracing cell borders based on WGA signal.
3
Immunofluorescence Analysis of Zebrafish Tissues
4
Immunofluorescence Analysis of Zebrafish Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
14-μm thick cryo-sections of zebrafish tissues were obtained by Leica CM-1900 cryostat. IF on cryo-sections was performed as previously described72 . The primary antibodies used were anti-GFP (Vanderbilt Antibody and Protein Resource), anti-collagen type-II (Rockland, #600-401-104), anti-Fibronectin (Sigma, #F3648), anti-β-catenin (Sigma, #C7207), anti-Matrilin (gifted by Dr. E. Kremmer), anti-Col1 (abcam, ab34710), anti-myosin (Developmental Studies Hybridoma Bank, #MF20). 4’,6-diamidino-2-phenylindole (DAPI, Molecular Probes) was used as a nuclear counterstain. Fluorescently labeled secondary antibodies used were either Alexa Fluor 488- & 555- (Life Technologies) or DyLight 550-conjugates (Thermo Scientific). Proteoglycan staining was performed using Alexa Fluor-555 conjugated wheat germ agglutinin (WGA, Life Technologies); muscle staining on sections was with Alexa Fluor 488-conjugated phalloidin (ThermoFisher, #A12379). Fluorescence imaging was carried out using AxioImager Z1 equipped with an Apotome (Zeiss) and an EC Plan Neofluar 100x/1.30 Oil objective.
5
Whole-mount Immunostaining Protocol for Zebrafish Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-mount immunostaining was performed as previously described with slight modification [40 (link)]. For Phospho-smad 1,5,9 antibody staining, embryos were fixed in 4% PFA, washed in PBS, boiled in sodium citrate solution for 2 minute and heated at 60°C for an hour for antigen retrieval. Embryos were blocked and incubated in primary antibody against Alcama (Zn5, Zebrafish International Resource Center) at a dilution of 1:250, Phospho-smad 1,5,9 (Cell Signaling, catalog no. 9511) at a dilution of 1:100, MF20 (Hybridoma bank) at a dilution of 1:50 or β-catenin (Sigma, catalog no. C7207) at a dilution of 1:500. Texas red-conjugated anti-mouse secondary antibody and Alexa Fluor 488 conjugated anti-rabbit secondary antibody was used at 1:100 dilution (Molecular probes/Invitrogen). Texas red or Alexa Fluor 647 conjugated phalloidin (Molecular Probes/Invitrogen) was used 1:100 dilution. Alexa Fluor 555 conjugated Wheat Germ Agglutinin (WGA, Molecular Probes), a lectin that binds to N-acetyl glycosamino glycan, was used at 1μg/ml concentration. TO-PRO-3 (Molecular Probes) was used at 1:1000 dilution for nuclear counterstain. To stain larvae and juvenile fish, hearts were dissected before incubating in the staining solution.
6
Drosophila Immune System Characterization
7
Imaging of S. aureus Infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on glass cover slips in a 12 wells plate followed by S. aureus infections as described above. Following gentamicin exposure to kill extracellular bacteria, cells were rinsed with PBS and fixed with 4% paraformaldehyde (Santa Cruz Biotechnology, United Kingdom). Cells were stained with 5 mg/L DAPI (Life technologies, United Kingdom) for nuclei staining and 5 mg/L Wheat Germ Agglutinin-conjugated Alexa Fluor 555 (WGA, Life technologies, United Kingdom) for membrane staining. Cover slips were mounted onto glass slides with FluorSaveTM (Calbiochem, United Kingdom). Images were visualized using a Leica SP5 confocal microscope using Advanced Fluorescence Software (Leica Microsystems, Milton Keynes, United Kingdom).
8
Quantifying Intracellular PHMB in Mac-T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mac-T cells were grown on glass cover slips in a 12 wells plate followed by S. aureus infections as described above. Following gentamicin exposure to kill extracellular bacteria, cells were rinsed with PBS and fixed with 4% paraformaldehyde (Santa Cruz Biotechnology, United Kingdom). Cells were stained with 5 mg/L DAPI (Life technologies, United Kingdom) for nuclei staining and 5 mg/L Wheat Germ Agglutinin-conjugated Alexa Fluor 555 (WGA, Life technologies, United Kingdom) for membrane staining. Cover slips were mounted onto glass slides with FluorSaveTM (Calbiochem, United Kingdom). Images were visualized using a Leica SP5 confocal microscope using Advanced Fluorescence Software (Leica Microsystems, Milton Keynes, United Kingdom). Sequential scan Z-stacks (130 slices 1024 × 1024) were compiled at a line average of 96, using Volocity® software. Three-dimensional (3D) Image Analysis Software was used for analysis and to produce 3D images. To quantify PHMB uptake into Mac-T cells, PHMB-FITC treated cells were incubated with 0.04% Trypan blue (Invitrogen, United Kingdom) in PBS for 15 minutes to quench membrane bounded PHMB-FITC, followed by flow cytometry (FACSBD machine and BDFACSDivaTM software, BD Bioscience) using the FITC filter. For each sample, 10,000 gated cells were analyzed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!