Additional Resources and Information
Department of Pathology, Children's Memorial Hospital:
Department of Pathology, Feinberg School of Medicine
Basic Histology Information:
Once a tissues is fixed, it must be processed into a form in which it can be made into thin microscopic sections. The usual way this is done is with paraffin, which is similar in density to tissue. Tissues embedded in paraffin can be sectioned at anywhere from 3 to 10 microns; we routinely section at 4 microns, unless otherwise indicated. The main steps in this process are dehydration and clearing.
Wet, fixed tissues (in aqueous solution) cannot be directly infiltrated with paraffin. First, the water from the tissues must be removed by dehydration. This is usually done with a series of alcohols. "Clearing" consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The most common clearing agent, and the one used in our lab, is xylene.
Tissues that come off the tissue processor are still in the cassettes and must be manually put into the blocks by a technician, who picks the tissues out of the cassette and pours molten paraffin over them. This embedding process if very important because the tissues must be aligned properly in the block of paraffin.
Once the tissue has been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome, which is simply a knife with a mechanism for advancing a paraffin block a standard distance. Plastic blocks are sectioned with glass or diamond knives. Paraffin blocks can be sectioned with thin disposable razor blades. Sectioning tissues is a real art and takes much skill and practice. It is important to have a properly fixed and embedded block or much artifact can be introduced in the sectioning (see below).
Tissue is snap frozen in a cold liquid or cold environment (-20 to -70 Celsius). Frozen sections are performed with a cryostat, which is simply a refrigerated microtome. The temperature inside the cryostat is approximately -20 to -30 Celsius. The tissue sections are cut, picked up on a glass slide, and are then ready for staining.
The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections. Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylene to alcohol to water. There are no stains that can be done on tissues containing paraffin. The staining process makes use of a variety of dyes that have been chosen for their ability to stain various cellular components of tissue. The routine stain is that of hematoxylin and eosion (H&E). Other stains are referred to as "special stains" because they are employed in specific situations according to the diagnostic need.
Frozen sections are stained by hand because this is faster for one or a few individual sections. The stain is a "progressive" stain in which the section is left in contact with the stain until the desired intensity is achieved.
Stained sections are coverslipped in order to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come. The stained slide must go through the reverse process that it went through from paraffin section to water. It is then taken through a series of alcohol solutions to remove the water, then through clearing agents to a point at which a permanent resinous substance beneath the glass coverslip can be placed over the section.
Some tissues contain calcium deposits which are extremely firm and which will not section properly with paraffin embedding owing to the difference in densities between calcium and paraffin. Bone specimens are the most likely type here, but other tissues may contain calcified areas as well. This calcium must be removed prior to embedding to allow sectioning. A variety of agents or techniques can be used.
Strong mineral acids, such as nitric and hydrochloric acids, are used with dense cortical bone because they will remove large quantities of calcium at a rapid rate. Unfortunately, these strong acids also damage cellular morphology and are not recommended for delicate tissues such as bone marrow.
Organic acids, such as acetic and formic acid, are better suited to bone marrow since they are not as harsh. However, they act more slowly on dense cortical bone. 10% formic acid is the best all-around decalcifier. Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time.
Artifacts in Histological Sections
A number of artifacts that appear in stained slides may result from improper fixation, the type of fixative, poor dehydration and paraffin infiltration, improper reagents, and poor microtome sectioning.
The presence of a fine black precipitate on the slides, often with no relationship to the tissue, suggests formalin-heme pigment has formed. This can be confirmed by polarized light microscopy because this pigment will polarize a bright white. Formalin-heme pigment is most often seen in very cellular or bloody tissues or in autopsy tissues because this pigment forms when the formalin buffer is exhausted and the tissue becomes acidic. This promotes the formation of a complex of heme (from red blood cells) and formalin. Tissues such as spleen and lymph node are particularly prone to this artifact. Making thin sections and using enough neutral-buffered formalin will help.
Tissues that are insufficiently dehydrated prior to clearing and infiltration with paraffin wax will be hard to section on the microtome, leading to tearing artifacts and holes in the sections. Tissue processor cycles should allow sufficient time for dehydration and final ethanol dehydrant solution should be at 100% concentration. Though alcohols make excellent fixatives for cytologic smears, they tend to make tissue sections appear brittle.
Bubbles under the coverslip may form when the mounting media is too thin. As it dries, air is sucked in under the coverslip. Contamination of clearing agents or coverslipping media may also produce a bubbled appearance under the microscope.