Tissue Preparation Methods

Primary fixation with some aldehyde(s) is the major determinant of fixation quality for ultrastructural studies. A wide variety of fixatives, including formaldehyde, glutaraldehyde, or mixtures of formaldehyde and glutaraldehyde—such as Karnovsky's fixative or McDowell's and Trump's 4F:1G (4% formaldehyde, 1% glutaraldehyde in a phosphate buffer)—are widely used with good results as described and illustrated in Chapter One of Dykstra and Reuss (2003).

Properly formulated formaldehyde, such as Carson's fixative (Carson et al. 1973) can produce good ultrastructural preservation, but some formulations used in histology laboratories allow the pH to drop below approximate neutrality (pH 7.2-7.4), which results in lower quality images. In the latter case, the ability to make a pathological diagnosis is often not compromised, but publication quality images are often not produced.

Pure formaldehyde made from paraformaldehyde powder (lacking preservatives like the methanol found in 37% formaldehyde stocks) is preferred for immunological studies. However, it does not produce the best preservation for conventional ultrastructural studies, particularly if post-fixation is eliminated, as is the case for immunological studies.

NTP prefers 4F:1G because it produces satisfactory results for both perfusion and immersion fixation of tissues, produces samples that section easily in histology laboratories, stains well with conventional histological stains (including periodic acid-Schiff's reagent), and produces excellent ultrastructural images. In addition, tissues have been successfully stored in 4F:1G in a refrigerator for decades (Dykstra and Reuss 2003; Dykstra 2010). The fixative is easy to formulate from inexpensive reagents and stores for up to 3 months in a refrigerator (Dykstra 2010). Plant tissues, animal tissues, protozoans, bacteria, viruses, and fungi have been successfully prepared using this fixative (see Dykstra and Reuss 2003).

The two major processing methods used to produce the samples in this Atlas are described below.

Automated Processing (utilized for large numbers of samples)

The samples were fixed in 4F:1G fixative. Processing was done with a Leica EM TP processor. Samples were rinsed with buffer, post-fixed in 1% osmium tetroxide in phosphate buffer, rinsed in water, and dehydrated in an ethanolic series, which culminated in acetone. The samples were then infiltrated with Poly/Bed 812 epoxide resin. After polymerization, the resulting blocks were trimmed, and semithin sections (approximately 0.5 micrometer thick) were cut, mounted on glass slides, and stained with 1% toluidine blue O in 1% sodium borate before being examined with a light microscope. After inspection by light microscopy to determine areas of interest, the blocks were trimmed for ultrathin sections (80-90 nm thick). These sections were subsequently placed onto 200 mesh copper grids and then stained with aqueous uranyl acetate and lead citrate as described in Reuss and Dykstra (2003). Digital images were captured with a Gatan Orius SC1000/SC600 attached to a FEICO Tecnai T120 transmission electron microscope.

Manual Processing (utilized for small numbers of samples)

Small numbers of samples were put through the same preparation protocols used for the machine-processed samples, except that the various fluid changes were performed manually with pipets.

Whole-body Perfusion

Some of the tissues were obtained from animals perfused, as shown in the example below, and stored in the fixative in the refrigerator for up to several months before further processing.

Immersion Fixation

Some of the organs were removed quickly from euthanized animals and immersed in 4F:1G fixative in a petri dish and cut so that at least one dimension was no more than 1 mm thick, because fixatives used for ultrastructural preservation generally do not penetrate more than 0.5 mm in an hour. If thicker pieces of the tissue are placed into fixative, the interior of the tissue blocks will be inadequately fixed for obtaining quality ultrastructural images.

Calcified Tissues

To prevent damage to diamond knives during the preparation of ultrathin sections, certain tissues that are calcified, like bone, are decalcified in Immunocal Formic Acid Bone Decalcifier from StatLab after primary fixation in 4F:1G. After approximately 12 hours in the Immunocal solution at room temperature, the tissues are rinsed in 3-4 changes of distilled water and then put back into 4F:1G for storage or put into 50% ethanol and processed further as described in the PolyBed 812 Resin Processing Schedule table below.

Processing Schedule

The following schedule for processing both immersion- and perfusion-fixed samples was used, whether manually or machine processed.

Carson F, Martin JH, Lynn JA. 1973. Formalin fixation for electron microscopy: a reevaluation. Am J Clin Pathol 59:365-373.

Dykstra MJ. 2010. Why 4F:1G fixative works for me. Microscopy Today. 18:50-53.

Dykstra MJ, Reuss LE. 2003. Biological Electron Microscopy: Theory, Techniques and Troubleshooting. New York: Kluwer Academic/Plenum Publishers.