Super fix rapid fixative solution

Whole rat eyes were fixed in SUPER FIX™ rapid fixative solution (Kurabo Industries, Osaka, Japan) and 3-μm paraffin sections were prepared from the fixed whole rat eyeballs in the usual manner [43 (link)]. The rat retinal tissue was observed in detail by hematoxylin and eosin (H.E.) staining. A microscope (Power BX-51, Olympus, Tokyo, Japan) was used for observation. The distance from the retinal ganglion cell to the outer granule layer (neural layer: inner plexiform layer, outer- and inner-nuclear (granule) layer) was calculated using Image J (NIH, MD, USA). The photographed area has a position of about 4–5 o’clock or 7–8 o’clock when the center of the cornea is at the 12 o’clock position in the sagittal section of the eyeball at approximately the middle part of the optic nerve and the peripheral part of the retinal nerve.

NIL was purchased from Tokyo Chemical Industry Co., Ltd. (Saitama, Japan). BAC was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan), and 0.4% Benoxil, 0.5% phenylephrine and 0.5% tropicamide were provided by Santen Pharmaceutical Co., Ltd. (Osaka, Japan). HPβCD and MC were kindly donated by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) and Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan), respectively. SUPER FIX™ rapid fixative solution was purchased from Kurabo Industries, Ltd. (Osaka, Japan), and the ELISA Insulin Kit was obtained from Morinaga Institute of Biological Science, Inc. (Kanagawa, Japan). Mannitol and streptozotocin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals used were of the highest purity commercially available. Wistar rats aged 7 weeks were provided by Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan). STZ rats were prepared by injecting Wistar rats intraperitoneally with 100 mg/kg streptozotocin twice on two consecutive days, and then housing them for two weeks after the last injection before use in this study. All experiments were performed in accordance with the ARVO resolution on the use of animals in research, and were approved by the Kindai University Faculty of Pharmacy Committee Guidelines for the Care of Laboratory Animals (project identification code KAPS-25-003, 1 April 2013).

On post-injection day 15, the mice were sacrificed by cervical dislocation. Their eyes were removed and fixed in SUPER FIX rapid fixative solution (Kurabo, Osaka, Japan). Fixed eyes were paraffin-embedded 5-μm sections were stained with hematoxylin and eosin (HE). Tissue section preparation and HE staining were entrusted to the Kyoto Pathology Laboratory. The images were captured using a BX-51 microscope equipped with a DP-25 CCD camera (Olympus, Tokyo, Japan).