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Basic Tissue Processing for Routine Light Microscopy

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Formaldehyde is a good immunoperoxidase technique as the tissues are fixed by cross-linkages formed by proteins and the cross-links does not harm the protein structure and the standard solution is 10% neutral buffered formalin. Glutaraldehyde is also used for fixation and the standard solution is 2% buffered glutaraldehyde.

This causes deformations in protein structure and penetrates very slowly but provides overall cytoplasmic and nuclear detail. Alcohols are usually not used for tissues as contain protein denaturants and cause brittleness. Oxidizing agents such as potassium permanganate is also not used frequently as they cross-link proteins and cause extensive denaturation. There is a number of factors which affect the fixation process such as buffering, penetration, temperature, volume, time interval and concentration of the solutions. Once the tissue has been fixed, they are processed into thin microscopic sections and embedded in paraffin. The technique of fixing tissues into paraffin is called tissue processing. There are two main steps in the processing which is dehydration and clearing. ‘Dehydration’ is a process of removing water from the tissues as wet fixed tissues cannot be directly infiltrated with paraffin. This is done by mixing alcohol (70%, 95% to 100%) with formalin. Once the tissues are dehydrated, the next step consists of removal of dehydrator with the substance that will be miscible with the paraffin. The common cleaning agent used for this step is xylene.
The next step involved is that the tissues are finally infiltrated with the embedding agent paraffin. ‘Infiltration’ is a process of impregnating the tissues with the embedding medium. This processed can be automated for a large number of tissues are processed. Automation consists of an instrument that moves through the tissues on a preset time. This activity can also be done with the help of a tissue processor by manually picking the tissues out of the cassette and pouring paraffin over them. This ‘embedding’ process is very important as the tissues need to be aligned properly in the paraffin. Plastics require special reagents which are expensive for dehydration and clearing and for these reasons, most of the embedding is done manually.

The next step involved is ‘sectioning’ which is done once the tissues are properly embedded. It is a process of cutting the tissues into sections such that they can be placed in the slide. This is usually done with the help of microtome which is a very sharp blade with advanced machines that can cut sections from 2 to 10 microns. The sections are cut and floated on a warm water bath that helps in removing wrinkles in the tissues and then they are picked up in the glass microscopic slide. These glass slides are warmed for 15 minutes to help the tissue section adhere to the slide. The next step is that the embedding process needs to be reversed so that the paraffin wax and water penetrate the tissue sections. The slides are ‘deparaffinized’ by cleaning the slides with xylenes to alcohol to water. Finally, the stained section is covered with a thin piece of plastic or glass which would prevent it from damaging and provide better quality. (Histotechniques)

Light Microscopy is the most common type used in the biological lab for tissue processing. A great advantage of light microscopy compared to acoustical microscopy is the fixation. sectioning and the staining techniques are optimized. Automated tissue processing has been achieved in the ‘technical’ tissue processor but it does not allow the light microscopy tissues to be processed in a fully automated environment. The studies revealed that treating the fluids in light microscopy has to be recirculated. Also, processing takes place in open or closed chambers which are not suitable for holding pressure or vacuum conditions. Technicon tissue processor also involves movement and dipping motions of the tissue specimens into open chambers and these movements made during processing tends to damage the specimens. Light microscopy also does not handle tissue processing effectively in the sealed system. (Light Microscopy processing method).

This has lead to the invention of utilizing dual containers for treating liquids including paraffin which are drawn to a sealed processing chamber. The tissues are confined to the closed chamber and remain stationary during processing. The programmed sequence is set which helps in fixation, processing and cleaning solutions in the closed chamber. Under this automated mode of operation, the solution pump in and out and replacing the next solution takes place in the preselected time sequence. While this invention is primarily in terms of light microscopy processing, elimination of cleaning steps, de-energization of heated paraffin will help in automated staining apparatus. Earlier, the microscopes used suffered poor resolutions and the addition of another lens resolved this. The process of tagging created glowing molecules which simplified the way for the researchers to obtain information. In summary, a series of improvement in light microscopy has lead to a new era in the field of biology. (Light Microscopy processing method)