Recent demonstration of in vivo imaging of glycans uses a bio orthogonal chemical reporter strategy of treating cells or organisms with alkin or azide tagged monosaccharides. Modified monosaccharides processed by the glycan biosynthetic machinery are incorporated into cell surface glyco conjugates, bio orthogonal alkin, or azide tags. Then allow covalent conjugation with fluorescent probes for visualization or with affinity probes for enrichment and glyco proteomic analysis.
This click chemistry based method allows for the rapid, non-invasive and robust labeling of alkin or azi detect glycans in zebrafish embryos and can be readily extended to visualize other classes of glycans in developing organisms. The main advantage of this technique over existing methods like lectin or antibody-based glycan detection methods is that it can be applied to dynamic in vivo imaging. Lectin and antibody-based imaging methods only provide a snapshot of the glycans at a particular time point.
They're difficult to implement in the context of the dynamic studies. In addition, labeling glycans with lectins or antibodies requires removal of the cells or tissues of interest from their native environment and fixing them before analysis. By contrast, labeling glycans using the bi orthogonal chemical reporter strategy overcomes these limitations and can be directly applied to non-invasive imaging of glycans in living systems such as zebrafish embryos and sea elegance embryos.
Using BTT Es mediated Azi Aine Cyclo edition, a biocompatible version of Qlik chemistry, robust labeling can be achieved within one to five minutes. Things treating the procedure will be data at both from my laboratory, Collect and transfer zebrafish eggs to a 35 millimeter Petri dish. Remove as much water as possible and then add one milligram per milliliter, pronase E in embryo medium to digest the corion after three to five minutes when bubbling of the corion may occur, merge the dish into a fish water filled beaker.
Gently transfer the eggs to the beaker by allowing the eggs to fall into the water. Rinse the eggs with fish water extensively such that most eggs will be released from their corion. Now using a fire polished pipette, transfer the coated eggs to agros coated Petri dishes filled with E three embryo medium Fill prepared injection dishes with E three embryo media and transfer in the coated eggs.
Next, prepare the needle and load with two microliters of injection solution containing 20 millimolar GDP Fuk all and either 5%lor, 5 94 dextrin as a tracer or 0.1%phenyl red loading dye in 0.2 molar potassium chloride as a negative control. Replace GDP FUCA with GDP Fuco in injection solution. Now break the needle and adjust the injection pressure and duration teal a one nanoliter drop.
Then inject the eggs with one nanoliter of the solution. Finally, transfer the injected eggs into agros coated Petri dishes filled with E three embryo medium and incubate at 28 degrees Celsius within three to four hours after fertilization. Remove the unfertilized eggs when the embryos reach the desired developmental stages, such as late gasa tissue segmentation and early larva.
Coat the base of a 96 well plate with aros. Add 92 microliters of embryo medium to each well followed by addition of four microliters ofor 4 88 azide from a 2.5 millimolar stock in water and two microliters of B-T-T-E-S copper sulfate six to one complex with a fire polished glass pastier pipette. Transfer less than five embryos into the well.
Continuing the click chemistry reagent add 2.5 microliters of freshly prepared 100 millimolar sodium ascorbate. To initiate the click reaction react for three minutes. Now add two microliters of 50 millimolar batho Cooper wine sulfonate a biocompatible copper chelator to quench the reaction and immediately dilute with 100 microliters of embryo medium.
Transfer the embryos to a glass petri dish and wash the treated embryos two times with 15 milliliters of embryo.Medium. Place a drop of ultra low melting point aros on a matte tech glass bottom micro well dish and transfer an embryo into the aros drop. Position the embryo and place the dish on ice for five minutes.
To solidify the agros drop gently add embryo medium to the dish until it covers the agros. Drop sequentially. Acquire fluorescence and brightfield images using a confocal microscope at five micron step intervals and construct composite figures.
Using image. J-B-T-T-E-S is atri amine based ligand that accelerates copper a c dramatically when coordinating with the NC two generated copper one and promotes the cyclo edition reaction rapidly in living systems without apparent toxicity. Here, zebrafish embryos are labeled via B-T-T-E-S mediated copper.
A a C at 9.5 hours post fertilization immediately following a three minute click reaction. Robust labeling of the GDPF all treated embryos is detected as expected. Only background fluorescence is detected for control embryos micro injected with GDP Fuco labeling and imaging can be performed anytime between 2.5 and 96 hours post fertilization.
These images are obtained at 24 and 48 hours post fertilization. Pulse chase. Using this strategy enable monitoring of the dynamic biosynthesis of sated glycans in which Alcon fuco produced at different time points are labeled with fluorophores of different colors.
This procedure can be combined with genetic mutants or morph based genetic suppression techniques to dissect the specific roles of individual glycan synthesizing enzymes during zebrafish.Embryogenesis. Though this method can provide insight into glyco biosynthesis. It can also be applied to other post translational modifications such as acylation at liquidation.
It can also be applied to the study of newly synthesized proteins.
