Comparison of the effects of the different fixatives on AQP-9 immunoreaktivity in some testicular cells

Determining the most suitable fixative for histomorphological and immunohistochemical studies of testicular tissue has periodically been a subject of interest in histopathology due to fixation-related issues. In immunohistochemical analyses, a key characteristic expected of a good fixative is its ability to preserve the target protein effectively and to enhance immunoreactivity robustly. This study compares the histomorphological effects of different fixatives on rat testicular tissue and their impact on the staining intensity of AQP-9 in testicular cells. In this study, fixatives routinely used in histopathology were employed: 10% neutral buffered formalin (10% NBF), modified Davidson's (mDF), Bouin's (BF), and Stieve's (SF). For each fixative, six tissue samples were analyzed, totaling 24 testicular tissue samples. Sections from previously prepared paraffin blocks were subjected to routine histological procedures, labeled with AQP-9 antibody using immunohistochemical methods, and analyzed through histomorphometric and immunohistochemical techniques. Histomorphometric measurements indicated that mDF and SF fixatives preserved the morphological integrity of rat testicular tissue and cellular details at similar, high levels. The 10% NBF exhibited the poorest performance in maintaining morphological integrity. Immunohistochemical analyses revealed that BF was the most effective fixative for revealing AQP-9 expression in germ and Sertoli cells of the seminiferous tubules, followed by mDF and SF. SF was the most effective fixative for revealing AQP-9 expression in Leydig cells of the interstitial area, with mDF and BF following in effectiveness. Additionally, 10% NBF demonstrated the poorest performance in exposing AQP-9 expression in both testicular areas. Our measurements suggest that for future histomorphometric studies of rat testicular tissue, mDF and SF should be preferred, while for immunohistochemical analyses of AQP-9 expression, SF, mDF, and BF are the preferred fixatives with comparable performance.