Near-isotropic, aligned volume electron microscopy (eFIB-SEM) of Mouse hippocampus (jrc_mus-hippocampus-3)

Sample: 2–3 month old male mouse, strain: C57BL/6J from Jackson Lab.

Sample Description: Tissue samples from the CA1 region of the mouse hippocampus were collected from 2–3-month-old male C57BL/6J mice. Samples were perfused with 3% paraformaldehyde, post-fixed, vibratome sectioned, and further fixed overnight in 1% PFA and 2% glutaraldehyde. 2 mm tissue punches were high-pressure frozen in 1-hexadecene, freeze-substituted with osmium tetroxide, uranyl acetate, and either 3-amino-1,2,4-triazole or imidazole, and embedded in Durcupan resin. The sample preparation was performed by Shu-Hsien Sheu. Imaging and post-processing were carried out by C. Shan Xu (Yale), Gleb Shtengel, and Harald Hess (HHMI/Janelia).
Protocol: Two datasets were acquired from the CA1 region of the mouse hippocampus using a hybrid fixation and staining protocol optimized for primary cilia visualization. 2–3-month-old male C57BL/6 mice were perfused with 3% paraformaldehyde and post-fixed in modified PFA buffer for 2 hours. Tissue was rinsed in 400 mOsM buffer and sectioned (100 µm) on a vibratome. Sections were fixed overnight in 1% PFA and 2% glutaraldehyde. 2 mm punches were high-pressure frozen in 1-hexadecene, freeze-substituted in osmium tetroxide, uranyl acetate, and either 3-amino-1,2,4-triazole or imidazole. Samples were embedded in Durcupan resin.

Contributions: Sample prepared by Shu-Hsien Sheu. Imaging and post-processing by C. Shan Xu (Yale), Gleb Shtengel, and Harald Hess(HHMI/Janelia).

Acquisition ID: jrc_mus-hippocampus-3
Voxel size (nm): 8 x 8 x 8 (x, y, z)
Data dimensions (µm): 60.9 x 81.28 x 45.07 (x, y, z)
Imaging start date: 2018-22-02
Imaging duration (days): 7
Landing energy (eV): 1200
Imaging current (nA): 2
Scanning speed (MHz): 2.083
Dataset URL: s3://janelia-cosem-datasets/jrc_mus-hippocampus-3/jrc_mus-hippocampus-3.zarr/recon-1/em/
Visualization Website: https://openorganelle.janelia.org/datasets/jrc_mus-hippocampus-3
Publication: Sheu et al., 2022; Xu et al., 2017