Vitreous Humor VEGF Quantification

Vitreous samples were collected using glass micropipettes. A micropipette puller is used to pull the tips of standard glass pipettes. Mice were anesthetized and the tip was inserted through the sclera 2 mm posterior to the limbus. Suction was activated by stepping on the foot pedal with a pump in reverse mode and vitreous was slowly aspirated, allowing collection of as much as 4 μL. Samples were immediately placed on ice, and the samples were centrifuged at 6000 rpm for 30 sec^{19 (link)}. 4 μL vitreous humor (from a single eye) was diluted in 6 μL RIPA buffer (Rockland Immunochemicals, PA, USA) containing protease inhibitor (Quartett, Berlin, Germany). VEGF protein levels of the vitreous humor samples were measured using a mouse VEGF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.