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CIL:54845*  Cite 

Transverse sections of 5 days post-fertilization wild-type zebrafish larva in the region of the anterior kidney

Technical Details

Brief description of sample preparation and SBEM imaging

5 d.p.f. zebrafish larva was prepared for SBEM. Larva was fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.15 M cacodylate buffer (CB, pH 7.4) at 4°C overnight. After removing the fixative, larva was treated for 15 minutes with 20 mM glycine in 0.15M CB, on ice to quench the unreacted glutaraldehyde. Then, larva was washed with 0.15 M CB and then placed into 2% OsO4/1.5% potassium ferrocyanide in 0.15 M CB containing 2 mM CaCl2. The larva was left for 30 min on ice and then 30 min at room temperature (RT). After thorough washing in double distilled water (ddH2O), larva was placed into 0.05% thiocarbohydrazide for 30 min. Larva was again washed and then stained with 2% aqueous OsO4 for 30 min. Larva was washed and then placed into 2% aqueous uranyl acetate overnight at 4°C. Larva was washed with ddH2O at RT and then stained with 0.05% en bloc lead aspartate for 30 min at 60°C. Larva was washed with ddH2O and then dehydrated on ice in 50%, 70%, 90%, 100%, 100% ethanol solutions for 10 min at each step. Larva was then washed twice with dry acetone and placed into 50:50 Durcupan ACM:acetone overnight. Larva was transferred to 100% Durcupan resin overnight. Larva was then flat embedded between glass slides coated with mould-release compound and left in an oven at 60°C for 72 h. The larva block was a Zeiss Gemini scanning electron microscope equipped with a Gatan 3View2XP and OnPoint backscatter detector. Images were acquired at 4.0 kV accelerating voltage with a 30 μm condenser aperture and 0.5 μsec dwell time; Z step size was 70 nm; pixel size was 9.5 nm; raster size was 25k x 12k, variable pressure under nitrogen. The volume was aligned using cross correlation, segmented, and visualized using IMOD.

Biological Sources
NCBI Organism Classification
Danio rerio
Cell Type
Whole larva
Keun-Young Kim
Sobhika Agarwala
Sebastien Phan
Saeyeon Ju
Ye Eun Kong
Guillaume A. Castillon
Eric A. Bushong
Mark H. Ellisman
Owen J. Tamplin
Digital Object Identifier (DOI)
Archival Resource Key (ARK)
Grouping This image is part of a group.
Image Mode