Different fixative methods influence histological morphology and TUNEL staining in mouse testes

• mDF/PFA fixation minimizes false TUNEL-positive cells in mouse testes.
• mDF/PFA fixation provides integrated morphologic details in mouse testes.

Society of Toxicologic Pathology has recommended mDF to fix testes since 2002. However, subsequent studies showed that false TUNEL-positive cells were observed in mDF-fixed testes. This study compared the effects of different fixation methods on histology and TUNEL staining in mouse testes. Results showed that fixation for 24 or 36 h in mDF provided better morphologic details in untreated testes, but markedly enhanced false TUNEL-positive staining. To optimize the fixation, testes were fixed using mDF for 6 h and then PFA for 18 h. Interestingly, fixation using mDF/PFA manifested better morphologic details, and rarely caused false TUNEL-positive cells in testes. Finally, we examined germ cell apoptosis in testes using mDF/PFA fixation in cadmium-treated mice. As expected, cadmium triggered germ cell apoptosis which was well visualized in the mDF/PFA fixed testes. Taken together, mDF plus PFA fixation not only minimizes false TUNEL-positive cells, but also provides integrated morphologic details in testes.