Encyclopedia of Experiments: Immunology
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Transcript
Dialyze 0.5 milliliters of the antibody using a dialysis cassette in 1 liter of conjugation buffer. After four hours, transfer the dialysis cassette to fresh buffer, and dialyze overnight. To set up a conjugation reaction, add 0.03 micrograms of IRDye 800CW per 1 milligram of antibody in a total volume of 500 microliters.
Incubate the mixture for two hours at 20 degrees Celsius. Then, purify the labeled conjugates by extensive dialysis against PBS. Estimate the degree of labeling by measuring the absorbance of the dye at 780 nanometers, and absorbance of the protein at 280 nanometers.
To develop mouse xenografts, gently, lift the skin of the animal and separate it from the underlying muscle layer. Slowly inject 100 microliters of cell suspension under the skin with a 26-gauge needle. Wait for a few seconds before taking the needle out so that basement matrix medium can form a semi-solid gel-like structure along with cells under the skin, which will prevent the mixture from coming out of the injection site.
To perform in vivo imaging, inject the dye-labeled antibody via the tail vein. After anesthetizing the mouse, check for the lack of response to pedal reflexes, and dilate the vein by applying warm water. Use a 1 CC insulin syringe with a 26-gauge needle to inject 25 micrograms of the labeled antibody in a volume of 100 microliters. As a negative control, label and inject the non-specific IgG isotype antibody which does not target cancer cells.
Perform in vivo imaging 8, 24, and 48 hours after antibody injections. In the imaging software, click Initialize. Confirm that the stage temperature is 37 degrees Celsius. In the control panel, set up fluorescence imaging through the imaging wizard, and set the excitation to 773 nanometers, and emission to 792 nanometers.
Transfer the anesthetized mouse into the imaging chamber and position it on the imaging field using the nose cone. When ready, click Acquire on the control panel for the image acquisition, and click Auto Expose. The generated image is the overlay of the fluorescence on the photographic image, with optical fluorescence intensity displayed in units of counts or photons.
