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Samples of tissues were also placed in cassettes and fixed in Bouin's solution for later histological processing (see Shields et al.
Firstly, the boiling shrimp sample was fixed to preserve its tissues in Bouin's solution.
After 30 min, cells were washed with phosphate-buffered saline (PBS) and fixed with Bouin's solution for 5 min.
The ovaries were removed, fixed with Bouin's solution, dehydrated in a graded ethanol-n-butanol series, embedded in paraffin and serially sectioned at 5 [micro]m thickness.
Ten of these abalone were sacrificed and immediately fixed in Bouin's solution for histological studies of gonad development and sex differentiation, using light microscopic observations of hematoxylin-eosinstained sections.
Furthermore, 4 abalone from each group were sacrificed and the gonads immediately fixed in Bouin's solution for histological examination to determine the developmental stage as defined by Sobhon et al.
For gonadal histology, 30 fish were fixed in toto for 24 hr in Bouin's solution and stored in 70% industrial methylated spirits (IMS) before processing for histology.
The edible part was removed from the shell, weighed to the nearest mg, fixed in Bouin's solution for 72 h, preserved in 70% alcohol, dehydrated in ethanol series and infiltrated with paraffin.