所属 関西医科大学 薬理学講座 職種 准教授
|発表タイトル||Changes in immunostaining of five elastic fiber components in Bruch’s
membrane and laser-induced choroidal neovascularization in mice
|会議名||ARVO 2016 Meeting|
|発表者・共同発表者||◎Mori Hidetsugu, Yamada Haruhiko, Takahashi Kanji, Akama Tomoya, Nakamura Tomoyuki|
Fibrillin-1, Developmental Arteries and Neural Crest Epidermal growth factor-like (DANCE), tropoelastin and Latent Transforming growth factor beta-Binding Protein-2 and -4 (LTBP-2 and -4) are essential proteins for elastic fiber formation. In this study, we analyzed the distribution of these five elastic fiber components in the elastic lamina of the Bruch’s membrane and laser-induced choroidal neovascularization (LiCNV) using immunofluorescence staining.
Twenty eyes of ten C57BL/6 female mice (8-10 weeks) were used. LiCNVs were induced in ten eyes by intense laser photocoagulation (532nm, 50μm, 350mW; GYC-2000®, NIDEK, Aichi, Japan). One week after photocoagulation, the eyes were enucleated and immediately frozen (-80℃). Eight micrometers thin-sliced sections were cut using a cryostat. Frozen sections were fixed with methanol at -20℃ for 5 minutes, washed with phosphate-buffered saline (PBS) for 5 minutes and blocked with 4% blocking agent for 20 minutes. They were incubated with primary antibodies (Fibrillin-1, DANCE, tropoelastin, LTBP-2 or -4) for 1 hour and were visualized with Alexa-488/546 and DyLight650 secondary antibodies for 1 hour at room temperature. After washing 3 times with PBS, all slides were mounted with ProLongGold® (Thermo Fisher Scientific, Waltham, USA) and observed under with a fluorescence microscope (BZ-X700®; KEYENCE, Osaka, Japan).
These five proteins (Fibrillin-1, DANCE, tropoelastin, LTBP-2 and -4) were expressed both in the elastic lamina of the Bruch’s membrane and inside LiCNVs in all eyes. The localization of these five proteins was similar. However, the signal intensity of DANCE was very faint compared to a positive control. Moreover, these five proteins were stained more intensely inside LiCNVs compared to the elastic lamina of the Bruch’s membrane.