Part A: Tissue Preparation
     2. LTP Physiology Protocol
          a.   Traditional Interface
     3. Acquiring Tissue Region
          a.   Vibroslicer VT 1000S
          c.    Stoelting Tissue Chopper
     4. Tissue Processing
          a.   Longhand
          b.   Embedding of Tissue
                  i.   Routine Embedding of Tissue
                  ii.   Re-embedding Tissue
                  iii.   Resurfacing Block

Part B: Microtomy
     1. Glass Knife Making
     2. Coating Grids
     5. Thick Sectioning
     6. Thin Sectioning
     7. Staining Grids
     8. Uranyl Acetate Recipe
     9. Lead Citrate Recipe
     10. Serial Sectioning

Part C: Electron Microscopy
     1. Alignment for TEM
          a.    2013 Nanoimaging Kuwajima et al. (PDF)
          b.    2013 PLoS ONE Kuwajima et al. (PDF)

Part D: Safety
     1. Supplemental Safety Protocols
          a.    Neutralizing the Aldehydes

          c.    Monitoring for Radioactivity

Overviews 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)
  • The Harris Lab Wiki features current protocols used by the lab on topics such as experimental design, EM image processing, and data analysis (3D Reconstruction).

Harris Lab papers (tissue processing):

  • Feinberg MD, Szumowski KM, Harris KM (2001) Microwave fixation of rat hippocampal slices. In: RT Giberson, RS DeMaree Jr. (eds.) Microwave Techniques and Protocols. Humana Press: Totowa, New Jersey. pp. 75-88. (PDF)
  • Jensen FE, Harris KM (1989) Preservation of neuronal ultrastructure in hippocampal slices using rapid microwave-enhanced fixation. J. Neurosci. Methods. 29:217-230. (PDF)

Harris Lab papers (EM imaging and Reconstruct):

  • Fiala JC (2005) Reconstruct: A free editor for serial section microscopy. J Microscopy 218:52-61. (PDF)
  • Fiala JC, Harris KM (2001) Extending unbiased stereology of brain ultrastructure to three-dimensional volumes. Journal of the American Medical Informatics Association. 8(1):1-16. (PDF)
  • Fiala JC, Harris KM (2001) Cylindrical diameters method for calibrating section thickness in serial electron microscopy. J Microscopy 202(3):468-472. (PDF)
  • Harris KM (1994) Serial electron microscopy as an alternative or complement to confocal microscopy for the study of synapses and dendritic spines in the central nervous system. In: Three-dimensional confocal microscopy: volume investigation of biological specimens (Stevens JK, Mills LR, Trogadis JE eds), pp 421-445. New York: Academic Press, Inc. (PDF)
  • Kuwajima M, Mendenhall JM, Lindsey LF, Harris KM (2013) Automated Transmission-Mode Scanning Electron Microscopy (tSEM) for Large Volume Analysis at Nanoscale Resolution. PLoS ONE 8:e59573. (PDF)
  • Harris KM, Spacek J, Bell ME, Parker PH, Lindsey LF, Baden AD, Vogelstein JT, Burns R (2015) A resource from 3D electron microscopy of hippocampal neuropil for user training and tool development. Sci Data Sep 1;2:150046. PMCID: PMC4555877. (PDFTables)
  • Tracing Synapses by P.H. Parker, a detailed informal tutorial on tracing synapses in Reconstruct (PDF).
  • Protocol for Ribosome Identification by L. Ostroff, a detailed tutorial on identifying polyribosomes in EM images (PDF).

Relevant publications from other labs:

  • Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A-138A. (PDF)
  • Karnovsky MJ (1971) Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. 11th annual meeting of the American Society for Cell Biology. New Orleans, Louisiana. (PDF)
  • Luft JH (1961) Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409-414. (PDF)
  • Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208-212. (PDF)
  • For retina: Leung IY, Sandstrom MM, Zucker CL, Neuringer M, Snodderly DM (2004) Nutritional manipulation of primate retinas, II: effects of age, n-3 fatty acids, lutein, and zeaxanthin on retinal pigment epithelium. Invest Ophthalmol Vis Sci, 45(9):3244-56. (PDF) (Note from Max Snodderly: "We used an osmotic agent to help preserve the RPE because the choroidal circulation is fenestrated and you need very large molecules to keep water from moving into the tissue and creating vacuoles.")