Animal studies were conducted in compliance with the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Duke University. In our standard protocol, C57Bl6/J mice (4–12 wk old; The Jackson Laboratory) were deeply anesthetized with ketamine/xylazine (100/10 mg/kg) and immediately transcardially perfused for 10 min with 15 ml fixative containing 2% paraformaldehyde, 2% glutaraldehyde, and 0.05% CaCl2 in 50 mM MOPS buffer, pH 7.4. Their enucleated eyes were postfixed in the same fixative for 1 h. A modification of this protocol included mouse perfusion with 10 ml heparin (1,000 U/ml) saline before paraformaldehyde/glutaraldehyde administration. Alternatively, we used the protocol used in the study by Chuang et al. (2007) (link). In brief, mice were sequentially perfused with 10 ml heparin saline administered for 2 min, 20 ml 4% paraformaldehyde/3.75% acrolein (Polysciences) in 0.1 M sodium phosphate buffer, pH 7.4, and finally 60 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer. Enucleated eyes were postfixed in 2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 1 h. A modification of this protocol included direct mouse perfusion with fixative, omitting the saline step. Upon the completion of animal perfusion, eyeballs were rinsed with PBS, cornea and lens were removed, the posterior eye cups were embedded in 5% agar (A1296; Sigma-Aldrich), and cut on a vibratome (Leica) at 100–200-µm sections.
A subset of experiments was performed with immersion-fixed eyecups. Mice were euthanized with CO2 and decapitated. Eyes were enucleated and eyecups immersed in 2% paraformaldehyde, 2% glutaraldehyde, and 0.05% CaCl2 in 50 mM MOPS buffer, pH 7.4, or 2% glutaraldehyde and 0.05% CaCl2 in 50 mM MOPS buffer for 1 h. Posterior eye cups were then embedded and sectioned as described in the preceding paragraph.
A subset of experiments was performed with immersion-fixed eyecups. Mice were euthanized with CO2 and decapitated. Eyes were enucleated and eyecups immersed in 2% paraformaldehyde, 2% glutaraldehyde, and 0.05% CaCl2 in 50 mM MOPS buffer, pH 7.4, or 2% glutaraldehyde and 0.05% CaCl2 in 50 mM MOPS buffer for 1 h. Posterior eye cups were then embedded and sectioned as described in the preceding paragraph.