Methods


Paraffin-Embedded Tissues

This is an appropriate medium for most routine histology. Fixed tissue pieces are dehydrated though a series of graded alcohols before clearing with xylenes. The final step in the processing is the replacement of the xylenes with molten paraffin. Tissue is then removed from the processing machine and is infiltrated with fresh molten paraffin, oriented, and allowed to cool and harden into a block. Tissue can then be sectioned on a microtome and slides produced.

What you need to do:

  1. Label the cassettes.
    Each cassette must be labeled with your initials and a unique identifying code. Cassettes must be labeled with a dark pencil, since the ink in most lab markers is soluble in alcohol or xylenes. The way you label the cassette determines the labeling of the slide. While you want to completely identify the specimen, the available space on the cassette and slide is limited, so you may have to develop a code to use consistently. We will be happy to help you with this, but here are two examples:
    1. Joe Doe is doing a complete necropsy consisting of 10 cassettes on each of 25 animals. To identify them, he labels them in this manner.
      JD 1 A through JD 25 J
      JD=his initials
      Number=animal number
      Letter=type of tissue in cassette (all A cassettes might be liver, all B cassettes urogenital, etc.)
    2. Sally Smith uses only the liver from her animals. She could use the above code (without the tissue letter) or she could use this type of code.
      SS 1-1
      SS=her initials
      First number=experiment number
      Second number=animal number
    Whichever method you choose to use to identify your cassettes, it should allow each of them to be unique. Your cassettes will probably be processed with cassettes from other investigators, so we need to be able to determine which cassettes belong to which investigator.
  2. Dissect the tissue.
    Most tissues process best when less than 4mm in thickness, 15 mm in width and 20 mm in length. This is also a manageable piece to section nicely. Often it is convenient to dissect directly into a cassette, but it is also possible to fix whole animals and dissect and cassette after fixation is complete.
  3. Choose the fixative.
    For routine work, the most popular fixative is buffered 10% formalin (10% solution of 37% formaldehyde solution in PBS). This works well for hematoxylin and eosin staining, special stains, and many immunohistochemistry and in-situ protocols. It is not necessary to go to the trouble to make a 4% paraformaldehyde solution for histology fixation, as this degree of purity is not required. Properly dissected tissue will be adequately fixed after 12 hours in formalin. After 24 hours, there is danger of overfixation, so the fixative should be replaced with 70% ethanol for storage and transport to the Histology Facility.
    If your protocol suggests another fixative, let us know what you are using so that we can help you to optimize it.
  4. Decide orientation.
    When tissue is ready for embedding in paraffin, it must be oriented. In some tissues, for example a piece of liver, this will not be an important consideration, but in tissues such as skin, proper orientation is essential. For example, if you are looking for the trigeminal ganglion in a 12 day mouse embryo, we need to know this to orient the embryo in a sagittal plane and section deep enough to reach this structure. Your may decide to embed your own specimen, or indicate on the requisition the desired orientation and we will do this for you.
  5. Determine the type of sectioning required.
    Routine histology involves sectioning one slide per block at 4 microns in thickness, and staining this slide with hematoxylin and eosin. Alternately, you may want to have extra slides cut to do special stains, immunohistochemistry, or in-situ hybridization. You may request these extra, unstained slides at the same time as the hematoxylin and eosin staining request or the blocks can be re-sectioned at a later date.

Frozen Sections

Many antigens and RNA do not survive routine histological processes. An alternative method requires tissue to be frozen and sectioned in a cryostat. Antigen preservation is always superior in fresh frozen tissue. Unfortunately, the drawback to frozen sections is the vastly inferior morphology of the tissue.

What you need to do

Tissue can be dissected, placed in a disposable mold which has been labeled as previously discussed, immersed in OCT (a freezing medium) and flash frozen in a bath of isopentane immersed in liquid nitrogen. Once again, orientation must be considered prior to freezing the tissue. Variations to this protocol include fixation in 10% formalin followed by immersion in various concentrations of sucrose and slow freezing on dry ice. We can help you determine the best method to demonstrate the particular antigen in a specific tissue.

Tissue should be stored at -80°C until it is sectioned.

Resin-embedded sections

The facility has available a Thermo automated microtome and tungsten carbide blade used in sectioning resin embedded tissue for the light microscope. The reagents used are glycomethacrylate resins that produce a firmer medium, allowing thinner (2 micron) sections to be cut. It is particularly useful for undecalcified bone. The disadvantages to this method involve the toxicity of the reagents and the limited stains that are compatible with the resin.

What you need to do

At this time, we do not process or embed tissue in resin, but we are able to section this material after you process and embed it. We can help you decide on appropriate stains for use with resin-embedded tissue.

Pathology Consultation

The Koch Institute has an affiliation with Dr. Roderick Bronson, a veterinary pathologist at Harvard Medical School. He is available to consult with investigators. Appointments are requested through iLab (Schedule Pathologist tab).

We hope this brief overview will be helpful in getting you started doing histology. If you have other concerns or issues you would like to discuss or if you would to observe the histology operation, please feel free to contact us at X8-8183.