Experimental Protocols

Overview of Harris Lab Methods

• Harris KM, Perry E, Bourne J, Feinberg M, Ostroff L, and Hurlburt J (2006) Uniform serial sectioning for transmission electron microscopy. J Neurosci 26(47):12101-12103. (PDF) (Supplemental movies) https://doi.org/10.1523/JNEUROSCI.3994-06.2006

• Kuwajima M, Mendenhall JM, Harris KM (2013) Large-Volume Reconstruction of Brain Tissue from High-Resolution Serial Section Images Acquired by SEM-Based Scanning Transmission Electron Microscopy. Methods Mol Biol (Nanoimaging: Methods and Protocols) 950:253-73. (PDF) https://doi.org/10.1007/978-1-62703-137-0_15

Previously used protocols

• A compilation of previously used tissue processing protocols

• Also see SynapseWeb here.

Notes on "purified water"

Unless otherwise noted, "purified water" mentioned in our protocols refers to the following:

• ASTM type I, double-distilled, WFI (water for injection), or equivalent. We use this.

  • ASTM type I water can be made with an appropriately maintained water filtration system (e.g., Milli-Q® Type 1 Ultrapure Water System)

Experimental Protocols

Animals

• Vaginal smear to figure out estrous cycle

• Urine Collection and Urinalysis

• Genotyping by Transnetyx

• Rapid decapitation and brain cryopreservation

• AAV injection

Electrophysiology

• Acute Hippocampal Slices

  • Notes on slicing-induced synaptogenesis

• Synchroslice

• MED64

• Microwave-assisted Chemical Fixation of Acute Hippocampal Slices

Perfusion-fixation

• Perfusion-fixation of rats and mice (can also be modified for marmosets)

EM Tissue processing

• Agarose Embedding and Vibraslicing Fixed Brain Tissue

• Long-term storage of chemically fixed vibratome-sectioned brain tissue

• Microwave-assisted Heavy Metal Staining and Epoxy Embedding of Fixed Brain Tissue

• en bloc only staining and epoxy embedding of chemically fixed tissue (70-250 µm thick tissue)

  • [Deprecated] en bloc staining and epoxy embedding of fixed brain tissue

• Imaging Tissue Blocks with the Leica Stereoscope

Ultramicrotomy

• Coating TEM Grids with Polyetherimide or Pioloform F

• Ultramicrotome and Serial Sectioning

• Toluidine Blue staining for semi-thin sections

• Post-section Staining of Ultrathin Sections with Uranyl Acetate and Lead Citrate

Electron microscopy - image acquisition

• tSEM Imaging

• TEM Imaging: see SynapseWeb (here and here)

• EM Tomography

Light Microscopy

• Coating microscope slides with gelatin

• Cresyl Violet staining

• Cresyl Violet Imaging

• Immunofluorescence labeling

Vibratome/Cryostat Sectioning

• Vibratome Sectioning

• Cryostat Sectioning (Drew Lab)

• Cryoprotecting Tissue

Others

• Shipping fixed brain tissue to the Harris lab for EM processing

• WESCOR VAPRO 5520 Osmometer

• Pre-embedding Immunohistochemistry

• Post-embedding Immunogold Labeling

Protocols under Development (restricted access):

• Osmium-Free Processing and Epoxy Embedding of Fixed Brain Tissue

• High-pressure Freezing and Freeze Substitution

• mAPEX2-related protocols

• Post-embedding immunogold labeling for GABA

• Using Zeiss Axio Observer 5 microscope

• Using Leica TCS SP5 confocal laser scanning microscope (Zemelman lab)

• Using Zeiss Axio Imager (Johnston Lab)

• Using Zeiss microscopes (Drew/Zemelman labs)