Ovaries collection and fixation
Six specimens of Astyanax lacustris were provided throughout 2013 by the Hydrobiology and Aquaculture Sector of Companhia Energética de São Paulo, in Paraibuna (CESP – Paraibuna), São Paulo, Brazil, for this and other procedures. Individuals were anesthetized and euthanized according to the protocol approved by the Committee on Ethics in Animal Experimentation of the Institute of Biomedical Sciences (ICB) of the University of Sao Paulo (USP), Proc. 2013/022.
After euthanasia, the ovaries were collected and left in phosphate-buffered saline (PBS pH 7,4) to avoid drying out. Then, the samples were prefixed for 1 hour in 4% paraformaldehyde and then fixed at 4 ºC in osmium tetroxide (2% OsO4). The use of a primary fixative and a post-fixative is sometimes necessary in order to combine the properties of the two chemical solutions, allowing for better imaging in the end.11 Thus, paraformaldehyde is a buffered solution that promotes a more subtle tissue fixation; when associated with osmium tetroxide, it ensures better tissue fixation, generating excellent results.11 After fixation, the ovaries were washed twice with PBS in order to remove excess of osmium, remaining in this same buffer overnight.