Orange g solution

The animal samples were collected and examined histologically. For hematoxylin and eosin (H&E) stain, sections were stained with Carrazi's hematoxylin solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 5 min and 1% eosin solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 30 s. To evaluate fibrosis, we used Masson trichrome (MT) stain. Briefly, after stained with Weigert’s iron hematoxylin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 5 min, sections were stained with following solutions; 0.75% Orange G solution for 1 min (Muto Pure Chemicals Co., Ltd., Tokyo, Japan), Masson B solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 20 min, and then aniline blue (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 6 min. For quantitative trichrome analysis, images of whole horizontal uterine section were electronically scanned at magnification of 200× and subsequently analyzed using ImageJ software. Additionally, for quantitative immunochemistry analysis, three random sections of trichromatic slides were also electronically scanned at magnification of 400× analyzed using ImageJ (version 1.32j) software (National Institutes of Health, USA http://rsb.info.nih.gov/ij/).