TEM Specimen Preparation

   Fixation: This is the most critical stage in the embedding process. Good results can be obtained with many fixatives, including neutral buffered formalin (NBF), but for optimal preservation of ultrastructural detail, we recommend 4% glutaraldehyde in 0.1M phosphate buffer for most solid tissue specimens. Whatever fixative is used, it is important that the specimen is placed in the fixative as soon as possible, and that there is sufficient fixative relative to the amount of tissue. If glutaraldehyde is used, the tissue should be no more than about 2 mm thick in at least one dimension. Formalin penetrates tissue faster than glutaraldehyde, but fixation will still be poor inside if tissue blocks are too large. See embedding reagents for reagent formulas.

   Storage: Tissue can be kept in NBF indefinitely, but tissue fixed in glutaraldehyde should be transferred to 0.1M buffer after 4 to 24 hours, and refrigerated at 4° C. For long-term storage (more than two or three weeks) glutaraldehyde-fixed speciments should be transferred to NBF.

   Embedding: The fixed tissue is washed with fresh buffer, and if necessary trimmed into smaller pieces for TEM embedding. Specimens are post-fixed with osmium tetroxide, and when appropriate, en-bloc stained with uranyl acetate. The specimens are then dehydrated using a series of ethanol solutions of increasing concentration. When dehydration is complete, they are transferred from 100% ethanol to propylene oxide, then to mixtures of propylene oxide and resin in increasing concentration. When resin infiltration is complete, the specimens are placed in labeled molds and placed in an oven for curing.

  Sectioning: The hardened blocks are trimmed with a razor blade until the tissue is exposed and excess resin is removed from the block face. Semi-thin sections for light microscopy are then cut from the block with glass knives, mounted on microscope slides, and stained with toluidine blue. The sections are examined with light microscopy, and if an appropriate area for electron microscopy if found, the block is trimmed down to the selected area, and ultra-thin sections are mounted on copper grids, and stained with uranyl acetate and lead citrate. An LKB Ultramicrotome is used for both semi-thin and ultra-thin sectioning. Ultra-thin sectioning is done with a diamond knife. After ultra-thin sections are made, mounted on glass slides, and stained with toluidine blue as before. These "after" sections allow the researcher to see exactly what area was sectioned for TEM, and are invaluable for correlating structures at the light and EM levels.

   Examination: The stained ultra-thin sections are examined with our JEOL JEM -100 CXII Transmission Electron Microscope. Micrographs are recorded on 3.25 x 4.25 inch negatives, which are printed in our darkroom or digitally scanned as needed.

Processing Cell Suspensions, Cultures, and Blood Fractions

   Processing of blood samples and cultured cells or organisms require special handling and fixation. Some samples can be centrifuged and pelleted, and treated as solid tissue. Other samples may require suspension in agar to avoid being dispersed during processing. To optimize your results, please contact us prior to processing and submitting your specimens.  

Reprocessing Tissue From Paraffin Blocks for TEM or Immuno-Bed

   Tissue that has been embedded in paraffin can be deparaffinized and processed for electron microscopy. This should only be done when unprocessed tissue is no longer available. Ultrastructural preservation is never as good after tissue has been processed through paraffin, and the procedure is not recommended unless the ultrastructural features of interest are relatively robust. With many specimens, however, quite satisfactory results can be achieved.

   Tissue that was originally embedded in paraffin can also be reprocessed for Immuno-Bed or JB-4 embedding. This procedure might be appropriate when thinner sections are needed, or if the tissue does not section well in paraffin, and a harder embedding matrix is necessary. The results are generally quite good.

   It is often possible to reprocess sections of paraffin embedded tissue that has been stained and mounted on glass slides for TEM. This should be considered only as a last resort, since the tissue has not only suffered from paraffin embedding and sectioning, but from the staining and mounting reagents as well. Since there is only 5 or 6 um of tissue available, results can not be guaranteed.