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.