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.
JD 1 A through JD 25 J
Letter=type of tissue in cassette (all A cassettes might be liver, all B cassettes urogenital, etc.)
First number=experiment number
Second number=animal number
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.
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.
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.
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.
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 may be arranged by contacting the histology staff.
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.