Serial section reconstruction of the optic nerve head in the area of transition between myelinated and unmyelinated axons the mouse obtained through serial section block face scanning electron microscopy. The location of the transition zone may be viewed on the image map (red box) associated with this data set.
Full resolution image description
Serial block face imaging SEM data set from the optic nerve head of the mouse in IMOD MRC format. Due to the size of the data file, you might have problems downloading the file. If so, please contact us, and we will ship the data to you.
3.0 spot size; 2.5 kV; high vacuum; 3x1 mosiac with 6k x 6k tiles; dwell time = 8 usec
Specimen preparation
Protocol used
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.The tissue washed with ddH2O at room temp 3x 5 minutes and placed in 2% osmium in ddH20 for an hour at room temp.The tissue washed with ddH2O at room temp 3x 5 minutes and placed in 1% aq. UA overnight in fridge.The next day, dissolve 0.066g of lead nitrate in 10 mL aspartic acid solution and adjust pH to 5.5 with 1N KOH. Place solution in 60 degree oven for 30 minutes. No precipitate should form.Take tissue from 1% UA and wash with ddH2O, 3x 5 minutes.Place tissue in the lead aspartate solution and leave in oven for 30 minutes. Wash tissue 3x 5 minutes with ddH2O and dehydrate and embed in Durcupan as described below.The tissue was dehydrated in EtOH (20, 50, 70, 90, 100, 100% for 10 minutes each) and then acetone (100% 3x 10 minutes). The tissue was placed into 50:50 Durcupan:acetone overnight. Tissue was placed in 100% Durcupan for 1 hour. Tissue was transferred to fresh Durcupan for 2 hours. Tissue was placed into 60 degree oven for 2 days and polymerized.
Imaging product type
Type
Serial section
Cutting plane
longitudinal
Description
Serial blockface imaging
Z resolution
70 nm
Notes
Gatan 3View on FEI Quanta
Citation Information
Keun-Young Kim, Eric Bushong, Mark Ellisman, Daniela Boassa, Masako Terada, Nicholas Marsh-Armstrong (2010) CCDB:8391, Mus musculus. CIL. Dataset. https://doi.org/doi:10.7295/W9CCDB8391