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.