Remember, the function of fixation is to preserve tissue in as lifelike manner as possible for histological examination.
As soon as tissue is removed from the body, pH within cells falls and lysosomal enzymes are released. Tissue rapidly starts to break down in process known as autolysis. In order to examine tissue histologically the process of autolysis need to be stopped as soon as possible after tissue removal. Failure to do so will allow autolysis to progress and will also cause diffusion of substances from their original locations.
There are a variety of methods for fixing tissue but the most common is chemical fixation by immersing pieces of tissue in a chemical solution.There are several thousand fixing solutions (due to the fact that no one fixing solution is ideal for all applications.) Fixation is most often acheived by cross linking the amino acid groups. Therefore, the most commonly used fixative is a buffered formaldehyde solution.
Fixation is generally accomplished by immersion of thin pieces (less than 5 mm thick) in the fixative for various periods of time, typically 24 hours.
At this point, the histologist has a lump of tissue in formaldehyde. That's not going to be very useful for cutting. In order to be able to cut sections thin enough for microscopic examination, tissue must be embedded in a medium (such as paraffin wax) hard enough when solid for such sections to be cut.
Processing requires:
1. The removal of water by use of a dehydrating agent such as graded ethanols from 70% up to 100% ethanol.
2. The use of an intermediary agent that is miscible with both alcohol and paraffin wax. This commonly xylene, a purified form of petroleum. This stage is also called "clearing" as the tissue becomes translucent during this step.
3. Infiltration with molten paraffin wax. This is normally at 58 to 60 degrees Centigrade and results in gradual removal of the xylene and infiltration of paraffin wax into the tissue. The paraffin penetrates into tissue components and even into the cytoplasm of individual cells.
4. Solidification of the tissue in a block of wax. The tissue is oriented for sectioning by placing in a mold and the paraffin wax allowed to solidify.
During fixation and processing most lipids are extracted (this is why adipose tissue looks like chicken wire). Some non proteinaceous substances will be lost in the fixing solution or during the subsequent process. Other substances such as glycogen may be retained as they are associated with proteins. In addition, due to the solvent action and to heat the tissue undergoes a certain amount of shrinkage. With soft tissues the shrinkage is generally in the order of 25 to 30%. Shrinkage often results in separation of components from each other such as separation of muscle fibers and separation of soft from hard tissue at interfaces. By now, you should be noticing this a lot in your example slides in lab!
Here's an example of shrinkage - the keratin layer in this high-power view of skin is separating from the underlying epidermal layers and from itself. The white spaces indicated by the arrows are artifact and not there in real life.
Sectioning of a paraffin-embedded tissue is very similar to frozen tissue sectioning. The solidified block of paraffin wax is fixed in a holder on the chuck on a microtome. The microtome allows a reproducible forward advancement of the block towards the knife that will slice a section from the surface of the block. Typical advancement is in the order of 5 to 10 microns.
As sections are cut, the lower edge of the block melts slightly allowing this edge to join with the trailing edge of the previous section thus forming a ribbon of sections. Such ribbons of sections as seen here may be stored for a short period of time.
During the sectioning a certain amount of compression occurs so that sections have the same width but are smaller from their upper to lower edges than the paraffin block from which they were cut.
Section mounting.
Sections are floated onto the surface of a warm water bath (usually 45 to 48 degrees F) where the paraffin section containing the tissue expands.
Sections are then collected and oriented on a glass slide and then allowed to dry.
Staining - it's business time!
Remember, most tissue components are of similar refractive indices and cannot be distinguished from each other unless we use phase contrast microscopy. The better way to differentiate tissue components is by the use of histological stains or histochemistry.
Most stains rely on binding to different components by virtue of some type of chemical bonding. We will deal with hematoxylin and eosin although the same general principals apply to many stains.
Sections on slides must first have the paraffin wax removed by soaking in xylene, followed by graded alcohols from 100% to 70% and finally into distilled water. Shown below are slides in the process of moving through the de-paraffinization process.
Sections are then stained in a solution of hematoxylin where the hematoxylin binds to acidic components. Next, we soak the sections in eosin which binds to basic components. The sections are then dehydrated, cleared in xylene and mounted in a transparent mounting medium. If done correctly, the stained and mounted slides will last for decades (as clearly demonstrated by your lab slide sets).
In a final well-stained preparation, nuclei are blue, and the cytoplasm stains a pale pink, (the cytoplasm of some cells may also be blue due to the presence of acidic components such as RER). Muscle cells stain bright pink, red blood cells bright red. Any traces of mineral will appear dark purple. This is especially noticeable in the partially mineralized enamel of developing tissue.
Decalcification (demineralization).
Embryos (while they do contain some mineral in developing teeth and bone)may be sectioned without removal of the mineral. However, tissues with considerable amounts of mineral such as bone, dentin and cementum must first have the mineral removed before paraffin wax sections can be prepared. This process is known as decalcification or more correctly as demineralization.
The tissue is first adequately fixed and then mineral is removed most often using dilute acids. During this process the tissue may show some changes due to the effects of the acid on components other than the mineral. Further hematoxylin-eosin staining then looks like this:
Bright pink staining of a tissue with very few cells is usually bone.
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