From Andrew Critch’s independent review of Nectome’s preservation of a rat brain under realistic end-of-life conditions.
Sample from a rat brain preserved using Nectome’s methods, then stored at -32°C for 12 hours.
Electron microscopy performed at the Berkeley EM Core.
Transmission electron microscopy
Brain samples were prepared as follows for EM processing (vibratomed) and post-fixed with 2.5% glutaraldehyde and 2.5% paraformaldehyde in buffer. Tissues slices were gentle cut into 1-2mm squared pieces using ultra-thin Personna razor blades (EMS, Hatfield, PA, USA). Tissue pieces were rinsed (3×; 5 min, Room Temperature) in 1x PBS and immersed in 1% osmium tetroxide with 1.6% potassium ferricyanide in 1x PBS for 1 hour. Samples were rinsed (3×; 5 min, RT) in buffer and briefly washed with distilled water (1×; 1 min, RT) before samples were then subjected to an ascending acetone gradient followed by pure acetone. Samples were progressively infiltrated with Epon resin (EMS, Hatfield, PA, USA) and polymerized at 60 C for 48 hours. A random block per condition was chosen for ultramicrotomy. Thin sections (90 nm) were cut using a Leica UC6 ultramicrotome (Leica, Wetzlar, Germany) and collected onto formvar-coated slot grids. Thick sections, 350 nm, were collected onto a PTFE-coated glass slide, heated to seal the section to the slide, and then stained with toluidine blue as a reference in light microscopy of the tissue. The grids were post-stained with 2% uranyl acetate followed by Reynold’s lead citrate, for 5 min each. Sections were imaged using a FEI Tecnai 12 120kV TEM (FEI, Hillsboro, OR, USA) and data recorded using a Gatan Rio 16 CMOS camera with Gatan Microscopy Suite software (Gatan Inc., Pleasanton, CA, USA). Montages were collected at 2900x magnification in 10 image x 10 image tile sets using SerialEM (bio3d.colorado.edu) and IMOD- eTomo (bio3d.colorado.edu) was used for montage reconstruction.
- Acetone : resin (with accelerator – BDMA) 2:1 for 1 hr
- Acetone : resin (with accelerator) 1:1 for 2 hr
- Acetone : resin (with accelerator) 1:2 overnight
- 90% resin (10% acetone) for 1 day
- Pure 100% resin (with accelerator) 3 X 1-2 hr [~ 6 hrs]
- Embed for polymerization in BEEM capsules
Acknowledgement
Thank you to Dr. Danielle Jorgens and Reena Zalpuri at the University of California Berkeley Electron Microscope Laboratory for advice and assistance in experiment planning, electron microscopy sample preparation, and data collection.