Animals were sacrificed on the sixteenth day and testes were removed under anesthesia, cut into two pieces each and kept in Bouin's fixative
for 48 hours.
The implantation sites were counted and fetuses were dissected out of the uterus and then fixed in Bouin's fixative
for 48 hours.
Fifteenth days after the start of the study, in sterile conditions, the left and right testes were removed by creating a gap in the lower abdomen, and the left testis was placed in Bouin's fixative solution for histological examination.
The left testis was removed from Bouin's fixative solution and tissue molding with paraffin and by microtome cut sections with a thickness of 5 microns were produced and stained with hematoxylin-eosin method.
hermafroditica, Bouin's fixative
penetrated tissue in less than 48 h, but, due to the high concentration of formalin (Bouin weak: 15% and strong: >20%), the specimens hardened and deteriorated at cellular level.
Two cover slips from each set of traps were preserved with modified Bouin's fixative
at a final concentration of 5% (Coats and Heinbokel, 1982), and four cover slips from each set were placed in small Petri dishes filled with water from the collection site for observation of live organisms with an inverted microscope (Invertscope, 25x magnification; Zeiss Corp.
For light microscopy, the specimens were fixed in Bouin's fixative
solution, processed routinely and the sections were stained with hematoxylin-eosin and Azo-Carmin to identify nerve fibers.
Whole fish were fixed in Bouin's fixative
and embedded in paraffin, and the gonad region was sectioned to 4 [micro]m, stained, and analyzed by light microscopy.
Gonads (from frogs at 1 month postmetamorphosis) attached to kidneys were fixed with Bouin's fixative
The uterus was immediately weighed, placed on absorbent cardboard in order to maintain the original in vivo orientation, fixed in cold Bouin's fixative
, and processed for histologic and morphometric examination.