TITLE: Electron Microscopy of Brain Tissue Samples
AUTHORS: Livia Hecke Morais, Mark Ladinski

[DESCRIPTION]
Protocol has been approved by the California Institute of Technology’s Institutional Animal Care
and Use Committee (IACUC).

[STEPS]
SECTION: Brain perfusion
1. Mice were anesthetized with 150 uL pentobarbital (Euthasol), and their hearts were punctured.

SECTION: Brain perfusion
2. Mice were perfused with 50 mL of 37°C PBS followed by 50 mL of 37°C 4% paraformaldehyde (PFA) at a flow rate of 6 mL/min.
SECTION: Brain perfusion
3.      
Brains were dissected and immediately placed in a cold (4°C) fixative solution of 3% glutaraldehyde, 1% paraformaldehyde (Electron Microscopy Sciences, EMS), 5% sucrose in 0.1M sodium cacodylate
trihydrate.
SECTION: Samples preparation
4.      
Tissue blocks were transferred to brass high-pressure freezing planchettes (Ted Pella, Inc.) prefilled with buffer containing 10% 70kD Ficoll (extracellular cryoprotectant).
SECTION: Samples preparation
5.      
Planchettes were placed into a high-pressure freezing machine (Bal-Tec HPM010) and ultra-rapidly frozen.
SECTION: Samples preparation
6.      
Planchettes with vitrified tissue samples were transferred under liquid nitrogen to cryotubes (Nunc) containing a frozen solution of 2% Osmium tetroxide (EMS), 0.05% uranyl acetate (EMS) in acetone.
SECTION: Samples preparation
7.      
Tubes were placed in a freeze-substitution machine (Leica Microsystems AFS2) and freeze-substituted as follows:
     -90°C for 72 hrs.
     Warm to -20°C over 24 hours.
     Hold at -20°C for 12 hours
     Warm to 4°C over 1 hour.
SECTION: Samples preparation
8.      
Samples, still within planchettes, were rinsed 3x with cold (4°C) acetone and brought to room temperature.
SECTION: Samples preparation
9.      Samples were infiltrated into Epon-Araldite resin (EMS) as follows:
     2:1 (Acetone:Resin) for 1 hour
     1:1 for 1 hour
     1:2 for 1 hour
SECTION: Samples preparation
10.      
Samples transferred into 100% resin and removed from the planchettes.
SECTION: Samples preparation
11.   Samples were allowed to infiltrate into resin for 24 hours, with gentle agitation.
SECTION: Samples preparation
12.      
Brain tissue samples were placed into resin containing accelerator (DMP30) and flat-embedded between two Teflon-coated glass microscope slides.  Resin was polymerized for 24-48 hrs at 60°C.
SECTION: Samples preparation
13.      
Embedded brain tissue blocks were examined with a dissecting microscope to select optimum regions.  These were excised with scalpel and reglued onto plastic sectioning stubs.
SECTION: Samples preparation
14.      
Semi-thin (170 nm) sections were cut with a ultramicrotome (Leica Microsytems UC6) using a diamond knife (Diatome, Ltd.).  Sections were collected onto formvar-coated copper-rhodium 1mm slot grids (EMS) and stained with 3% uranyl acetate and lead citrate.
SECTION: Imaging
15. Grids were imaged with a transmission electron microscope (Thermo-Fisher Tecnai T12-G2, 120k eV) equipped with a Gatan US1000 2k x 2k CMOS camera.  Large-area montaged images of brain regions were collected automatically using the SerialEM software package.
SECTION: Imaging
16.      
Images were subsequently aligned and analyzed using the IMOD software package and AIVIA image analysis software (version 10.5.0; Leica Microsystems) with pixel classifier machine learning software to identify mitochondria.