Eyes were marked at the nasal limbus, enucleated, briefly washed in ice cold PBS, and immediately embedded in tissue freezing medium (Leica Biosystems, 81-0771-00). 12- m-thick sections were prepared using a Leica cryostat. For COX enzymatic histochemistry, sections were incubated with COX detection solution (1.6 mM DAB, 1.25 mg/ml cytochrom C, 10 l catalase in PBS, pH 7.0) for 45 min at 37 C. For SDH enzymatic histochemistry, sections were incubated with SDH detection solution (1.55 mM nitrotetrazolium blue chloride, 0.13 mM sodium succinate, 0.2 mM phenazine methosulfate, 0.1 mM sodium azide in PBS, pH 7.0) for 25 minutes at 37 C. Sections were dehydrated in a series of ethanol solutions of increasing concentrations up to 100% and immersed in xylene. Coverslips were mounted with Cytoseal (Thermo Scientific, 8312-4). Bright-field microscopy images were acquired with a Zeiss microscope (Axioplan 2), processed and analyzed with ImageJ (National Institutes of Health). Pixel intensity profiles through the PS were obtained using the profile plots tool controlled by the line tool in ImageJ.
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