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)
- 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
- Synchroslice
- MED64
- Microwave-assisted Chemical Fixation of Acute Hippocampal Slices
Perfusion-fixation
- Perfusion-fixation of rats and mice (can also be modified for marmosets)
- A technical overview on cross-linking fixatives
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
- 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
- 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)
