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*CIL – Cell Image Library accession number. Please use this to reference an image.

CIL:54845*  Cite 
Description

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
Attribution
Names
Keun-Young Kim
Sobhika Agarwala
Sebastien Phan
Saeyeon Ju
Ye Eun Kong
Guillaume A. Castillon
Eric A. Bushong
Mark H. Ellisman
Owen J. Tamplin
Citation
Digital Object Identifier (DOI)
doi:10.7295/W9CIL54845
Archival Resource Key (ARK)
ark:/b7295/w9cil54845
Grouping This image is part of a group.
Imaging
Image Mode
SBEM