CIL:40262, Mus musculus, CNS neuron (sensu Vertebrata). In Cell Image Library

The animal was perfused with Ringer's followed by 2% PFA / 2.5% glutaraldehyde. The tissue was postfixed in the same fixative for 2 hours on ice. The eye was enucleated and the optic nerve head was dissected for sagittal longitudinal section.

The tissue was placed into solution containing 3% potassium dichromate and 2% osmium tetroxide in ddH2O. The samples were allowed to sit at room temp for 3 hours.

The tissue washed with ddH2O at room temp 3x 5 minutes and placed in filtered thiocarbohydrazide(TCH) solution(0.1 g TCH in 10 mL ddH2O) for 30 minutes at room temp.

After washes with ddH2O at room temp (3x 5 minutes) tissue was placed in 2% osmium in ddH20 for an hour at room temp.

Another series of washes (3x 5 minutes) was followed with 1% aqueous uranyl acetate overnight in fridge.

The next day, 0.066g of lead nitrate was dissolved in 10 mL aspartic acid solution and adjust pH to 5.5 with 1N KOH and incubated at 60° C for 30 minutes taking care that no precipitate formed.

Tissue was removed from 1% UA, washed (ddH2O, 3x 5 minutes) and placed in the lead aspartate solution, 60°C, 30 minutes.
After washes, tissue was dehydrated and embedded in Durcupan according to standard protocol.

This image was acquired at 1200X using a Gatan 3View system on FEI Quanta microscope.