1 M phosphate buffer solution) for 8-12 hours and their secondary fixation were done in 2 per cent osmium tetraoxide
for 2 hours.
The washed tissues were then allowed to undergo post-fixation with 1% Osmium tetraoxide
for 2 hours at 4[degrees]C, dehydrated with graded ethanol (30%, 50%, 70%, 90% and 100%) and dried with liquid C[O.
Then, they were post-fixated with 1% osmium tetraoxide
Samples were post-fixed in 2% aqueous solution of osmium tetraoxide
for 4h, then dehydrated in a series of graded alcohols and finally dried to a critical drying point with a Critical Point Dryer unit.
Tissues were transferred to a 1% solution of osmium tetraoxide
prepared with Millonig's, fixed for 2 hours, and then washed two times with Millonig's.
5% solution of glutaric aldehyde on the phosphate and cacodylate buffer, finished fixing with 1% solution of the osmium tetraoxide
after dehydration in the alcohol-acetone, then dried with method of critical point in the apparatus HCP-2 and were pulverized with the gold in the apparatus IB-2.
After fixation, the cells were covered with 1% osmium tetraoxide
(OsO4) in 0.
Specimens fixed as above were postfixed with 1% osmium tetraoxide
in 30 mmol [l.
The examined samples were first cut under cryogenic conditions with a Leica Ultracut microtome with a diamond knife to a thickness of about 70 nm and tinted with osmium tetraoxide
Washing in several changes of buffer for 30 minutes was then followed by post fixation in 1 percent osmium tetraoxide
in cacodylate buffer for 4 hours at room temperature.
4 at 4[degrees]C during 3 hours; they were washed with the same solution during 10 minutes and were then post-fixed in osmium tetraoxide
at 1% in buffer cacodylate during 1 hour.
5 % gluteraldhyde and 200 ul of a 1% osmium tetraoxide
each of which was suspended in the sodium/magnesium buffer at pH 7.