Prefer solution

We prepared the sections according to the method we described previously^{12 (link)}. Prefer solution (Anatech Ltd, Battle Creek, MI) was used to fix the mouse eyes for 15 min at room temperature, followed by 70% ethanol overnight. The tissue was paraffin-embedded, and 5-μm-thick sections were cut and mounted onto slides. These sections were subjected to immunohistochemistry. A Nikon Eclipse E800 microscope equipped with a digital camera was used to examine the antibody-labeled complexes. Metamorph (Universal Imaging, West Chester, PA) image analysis software was used to capture images under identical microscope and camera settings.

Mice were euthanized by CO₂ asphyxiation and the eyeballs were placed in Prefer solution (Anatech Ltd, Battle Creek, MI) for 15 min at room temperature, followed by 70% ethanol overnight. The tissue was paraffin-embedded, and 5-μm-thick sections were cut and mounted onto slides. Sections were deparaffinized in 2-3 changes of xylene (10 minutes each) and hydrated in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each, and then rinsed in distilled water. The slides were washed three times in 1X PBS containing 0.1% Triton-X 100, blocked with horse serum for 1 h, and primary antibody was added overnight at 4°C. For fluorescent detection, slides were incubated with a mixture of Texas-red-anti-mouse and FITC-anti-rabbit antibodies (Vector Laboratories, Burlingame, CA), each diluted 1:200 in PBS with 10% horse serum. After incubation for 1 h at room temperature, the slides were washed with PBS and cover-slipped in 50% glycerol in PBS. Antibody-labeled complexes were examined on a Nikon Eclipse E800 microscope equipped with a digital camera, and images were captured using Metamorph (Universal Imaging, West Chester, PA) image analysis software. All images were captured using identical microscope and camera settings so that intensities of the digital images reflected antibody binding.

Prefer solution (Anatech Ltd, Battle Creek, MI) was used to fix the mouse eyes for 15 min at room temperature, followed by 70% ethanol overnight. The tissue was paraffin-embedded, and 5-μm-thick sections were cut and mounted onto slides. These sections were subjected to immunohistochemistry. A Nikon Eclipse E800 microscope and Olympus MVX10 macroscopic imaging system for high resolution equipped with a digital camera was used to examine the antibody-labeled complexes. Metamorph (Universal Imaging, West Chester, PA) image analysis software was used to capture images under identical microscope and camera settings.