Methods for wholemount tissue preparation have been described in detail previously58 (link). After the animal was euthanized, its eyes were enucleated and placed in bicarbonate-buffered Ames solution (Ames; Sigma, A1420), equilibrated to pH 7.4. It has been shown that variations in the O2 level in brain tissue can affect functional hyperemia5 (link). To reduce any discrepancy, O2 level was maintained between 19% and 24%, checked with an oximeter (WPI ISO2-D) in the chamber. After dissection of the eyes, cornea, iris, and lens were removed. The retina was dissected into four equal quadrants and attached photoreceptor surface down on a modified Biopore Millicell filter (Millipore). This preparation was transferred to a recording chamber and bathed (1 ml/min) with Ames. Pharmacological agents were also prepared in Ames. All experiments were performed at a near physiological temperature of 32 °C.
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