Inclusion in DMSO, cryofracture and maceration
On the following day, samples were washed twice with distilled or deionized water, and subsequently immersed in dimethylsulfoxide (DMSO) at different concentrations. For the ovaries, the following times were standardized: in DMSO 25% for 30-40 minutes at 4 ºC, and in DMSO 50% for 60 minutes at 4 ºC. Once this preparation was complete, the samples were fractured in 50% DMSO cooled by liquid N2 and then immersed in 50% DMSO until they returned to room temperature. Subsequently, the samples were washed in distilled or deionized water for 2 hours (15 changes of 5 minutes each) at 4 ºC.
Finally, fractured samples were taken for maceration in 1% osmium at room temperature. The maceration was carried out in a shaker, with gentle movements, for 15 hours. The post-cryofracture maceration process enables the emptying of organelles and other cellular structures, allowing visualization of the plasmalemma and its specializations.12 This is an especially interesting situation when one considers the amount of yolk vesicles that occupy the interior of oocytes in fish. In addition, this process presents an inverse dependence between time and temperature, as for lower temperatures, as is the case of this study, it is necessary that the samples remain in maceration for a longer time.12 In this case, the use of lower temperatures and longer time is advantageous, as it allows a more delicate manipulation of tissue components.