On the day of the injection, after confirming the depth of general anesthesia, anesthetize the cornea with one drop of 0.5% tetracaine, and dilate the pupil with one drop of 2.5% phenylephrine. After dabbing off excess liquid, add one drop of 5% betadine to the eye surface, and surrounding hair to decrease the risk of endophthalmitis.
After two to three minutes, remove the betadine, and cover the eye with 0.3% hypromellose gel to prevent dryness under anesthesia and cataract formation. Next, load a 10-microliter syringe with the antigen and fluorescein mix. Then, place the mouse in a prone position on the platform and use the right and left ear bars to fix the mouse head.
Position and orient the mouse under the scope so that the superior nasal aspect of the right eye is visible. Then, using a 30-gauge needle, displace the eyelashes, expose the sclera, and visualize the limbus and the radial blood vessel.
Next, using a sterile 30-gauge needle make a guide hole in the sclera 1 to 2 millimeter posterior to the limbus. Then, insert the 34-gauge needle attached to the injection holder into the eye through the guide hole at an angle that will avoid the lens, but place the needle tip into the vitreous cavity.
Using a micro syringe pump controller, carefully inject 1 microliter of the M tuberculosis extract into the vitreous cavity. In case of consistent reflux, increase the injection volume to 1.5 microliters to ensure adequate dose delivery. Confirm the intravitreal placement by visualizing a greenish reflex in the eye, and after ten seconds, remove the needle from the eye.
