Mice were perfused with ice-cold phosphate-buffered saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer and the eyes were rapidly enucleated. The right eyes were embedded in paraffin wax, cut into 7 μm thick sections, and stained with hematoxylin and eosin. The left eyes were embedded in Tissue-Tek OCT Compound (Sakura Finetechnical, Tokyo, Japan) and frozen. Retinal sections of 10 μm thickness were cut on a cryostat at −20 °C and examined by immunostaining using antibodies against RNA-binding protein with multiple splicing (RBPMS; 1:500; ABN1376; Merck Millipore, Burlington, MA, USA) and osteopontin (1:1000; AF808; R&D, Minneapolis, MN, USA).
Stained sections were examined using a microscope (BX51; Olympus, Tokyo, Japan) equipped with Plan Fluor objectives (Olympus) connected to a DP73 camera (Olympus). Images were processed and viewed using a DP manager software (v2.2.1.195; Olympus). For quantification, the number of total cells, RBPMS-positive or osteopontin-positive cells in the ganglion cell layer (GCL) was counted from one ora serrata through the optic nerve to the other ora serrata [15 (link)] in three sections per mouse. The number of mice examined is shown in each figure legend.
Stained sections were examined using a microscope (BX51; Olympus, Tokyo, Japan) equipped with Plan Fluor objectives (Olympus) connected to a DP73 camera (Olympus). Images were processed and viewed using a DP manager software (v2.2.1.195; Olympus). For quantification, the number of total cells, RBPMS-positive or osteopontin-positive cells in the ganglion cell layer (GCL) was counted from one ora serrata through the optic nerve to the other ora serrata [15 (link)] in three sections per mouse. The number of mice examined is shown in each figure legend.