We've had a lot of dehydrated, exhausted bats.
The bone was decalcified in formic acid (29 g citric acid, 18 g trisodium citrate dihydrate and 100 ml formic acid, with distilled water added to yield a total volume of 1000 ml), dehydrated and embedded in paraffin.
Slides with dried frozen sections were placed in a preincubation solution composed of distilled water (475 mL), dehydrated potassium acetate (2.45 g), and calcium chloride (1.30 g) at pH 4.5 for 2 minutes; slides were subsequently rinsed in Tris buffer.
Briefly, 1 ml of each sample was dehydrated by adding 10 ml chilled acetone.
The fixed washers were rinsed with 4 mL of deionized water five times and dehydrated at room temperature in seven steps by placing the washers for 5 min in 4 mL of 50, 70 (twice), 85, 95 (twice) and 100% ethanol respectively.
The final extract was washed with 1 mL of 5% sodium chloride and then dehydrated with sodium sulfate and concentrated to 5 mL for further cleanup.
Biological samples were prepared on a 0.03 μm poly ether sulfone) membrane filter (Sterlitech) by fixation with 2.5% glutaraldehyde (diluted from 25%) in PBS for 4 h at room temperature, then serially dehydrated with 10 mL of H2O (twice), 25% ethanol, 50% ethanol, 75% ethanol, 80% ethanol, 90% ethanol, and 100% ethanol (twice).
Here, the seed solution was prepared by dissolving 10 mM of zinc acetate dehydrate and 1 mL of sodium dodecyl sulfate solution in 50 mL of ethanol.
The samples were incubated in the staining solution for 8 h at 37 °C, after which they were cleared of chlorophyll, dehydrated, and placed in the clear solution (chloral hydrate/water/glycerol = 8 g/4 ml/2 ml) for microscopic observation.
