Immunohistochemistry (IHC) of myelin basic protein (MBP)

The six pairs of control and experimental eyes were sectioned (4 µm) and the sections were flat-mounted on poly-L-lysine-coated slides. The sections were dewaxed and blocked with 5% goat serum (G9023; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) diluted 1:20 in PBS, at room temperature for 20 min, then exposed to mouse monoclonal anti-MBP (1:500; cat. no. ab24567; Abcam, Cambridge, MA, USA) prepared in PBS (0.1 M, pH 7.4) for 1.5 h at 37°C. All specimens were subsequently given three washes over 15 min in a 4°C PBS wash solution. Slides were then incubated at room temperature with the secondary antibody: Supervision Anti-Mouse Detection Reagent (undiluted; cat. no. D-3001; Kangwei Biotechnology, Co., Ltd., Beijing, China), for 30 min. Specimens were washed three times over 15 min in a 4°C PBS wash solution and the staining was revealed using 0.05% diaminobenzidine at room temperature for 30 sec. Slides were subsequently mounted in neutral resin.

Myelin and retina were observed with an optical microscope at magnifications of ×10 20, and 40. Images were captured with strictly controlled parameters. Images were analyzed using the Q500IW image analysis software (Leica Microsystems GmbH, Wetzlar, Germany). Images were transformed into black-and-white images. The light intensity was set to a range of 0–100; full penetration of the light was given a value of 100, whereas full light obstruction was given a value of 0. The difference in light intensity between background and myelin was regarded as an indirect representation of the MBP content.