For the histopathological analysis of these organs, the samples were fixed in 10% buffered formalin, and the tissues were processed by conventional techniques in 5-mm-thick paraffin slices.
Following cryoprotection in 25% sucrose buffer overnight at room temperature, the tissues were embedded in Tissue Tek O.C.T compound and rapidly frozen in -80°C.
The assay buffer was decanted and the tissues were bleached in 95% EtOH and photographed.
Then, recently prepared buffer solution was added and the tissues were then homogenized mechanically, sonicated (3 pulses of 10 seconds, 50% amplitude), and finally centrifuged (13000 g, 7′ to 4°C).
After rinsing in same buffer the tissue was dehydrated through a series of graded ethanol to propylene oxide, infiltrated and embedded in epoxy resin and polymerized at 70°C overnight.
After rinsing in phosphate buffer the tissue was trimmed into 2 mm thick transverse slices.
After rinsing in cacodylate buffer, the tissue was postfixed in 1% cacodylate buffered osmium tetroxide for 2 h at room temperature, then dehydrated in a graded series of ethanol, quickly transferred to propylene oxide and embedded in Epon-Araldite.
The tissues were fixed in 10% buffered formalin and were processed using a tissue processor.
The tissues were fixed with 10% buffered formalin, and were then embedded in paraffin.
