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The reaction was stopped with 10% acetic acid and washed with water.
In the present study, however, the red/green fluorescence ratio significantly increased in zona-penetrated spermatozoa compared with zona-binding ones, following treatment with acetic acid.
This method uses a mixture of anhydrous acetic acid and sulphuric acid, which is heated to prepare pollen for examination under a light microscope.
Trematodes were fixed in glacial acetic acid and pressed between two slides, and preserved in 70% ethyl alcohol.
When half of the thiocyanate solution was added, 25 ml of glacial acetic acid was dropped and the solution was stirred for two more hours.
The harvested eyes were placed in cold acetic acid/methanol solution overnight followed by paraffin embedding using standard procedures.
A very small volume of fixative (methanol 3 : acetic acid 1) was poured into the hypotonic solution.
A freshly prepared mixture of 3 volumes methanol: 1 volume acetic acid was slowly run onto the pellet and poured off; this was repeated twice.
If cytoplasm remained, it was removed by spreading one drop of another fixative, a mixture of methanol and acetic acid (3:1).
The reaction was stopped in 10% acetic acid.
Final preparation was carried out with a needle and diluted acetic acid.
Heptane-containing solutions with identical concentrations to those of the acetic acid and sodium hydroxide treatments were utilized with the same protocol as above.
The repeated immersion technique was used (as in the bleach treatments described previously) with various total duration times of heptane and acetic acid.
The stain was then differentiated with acetic acid and 70% ethanol to ensure staining of only the ganglion cell layer.
Twenty-four hours after insemination, the oocytes were fixed in methanol/acetic acid (3 : 1, v/v) at room temperature and stained with 1% aceto-orcein.
