Tissue Sectioning: microtome techniques, procedures and protocols
The preparation of high-quality tissue sections for histopathology requires compliance with specific techniques, procedures and protocols as well as skill and experience.
The quality of the cut is strongly influenced also by the previous steps:
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Fixation
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Processing
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Embedding
Fixation is the most important step in the histological sample preparation procedure. The duration of fixation is important and varies depending on the nature of the biological sample under examination, but above all on the basis of the thickness of the piece.
Tissues fixed for an insufficient time (underfixed) will not be adequately protected from the action of the reagents and exposed to greater artifacts. Prolonged fixation (overfixed) can compromise tissue reactivity, especially with regard to subsequent immunohistochemical investigations.
TISSUE EMBEDDING WITH PARAFFIN
At the end of the processing the sample is inserted into molds and covered with liquid paraffin. Then, the cassette with the identification number of the patient and additional paraffin is placed on the mold. The mold is then laid on a cold surface in order to favor its solidification and subsequent creation of the block.
TYPES OF MOLD
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Paper mold
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Metal mold
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Dimmock mold
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Disposable mold (plastic)
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Metal or plastic mold used for ring cassettes (F) or standard cassettes (G)
MICROTOME BLADE ORIENTATION
SPECIMEN ORIENTATION
The orientation of the cassettes is very important and can have a high impact on the cut and on the possibility of obtaining a "strip" of sections.
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In most laboratories all the cassettes are placed in the clamp in the same way (eg. with the label on the left for east-west orientation, or on the top for north-south orientation). Therefore it is necessary to consider the orientation of the sample with respect to the blade already in the embedding phase. There are different opinions regarding the optimal orientation for certain types of samples. Diapath Galileo Series 2 microtomes are also equipped with a precision orientation system with commands that allow you to easily find the zero position or a measurable variable on the x/y axis. This function is very useful when cutting blocks prepared in other laboratories or on other microtomes, where precise alignment is required to avoid loss of tissue
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In the routine of cutting several blocks, it is important that the specimen holder is set to zero on both axes before starting sectioning.
RETRACTION
Retraction is a feature that offers advantages in the sectioning phase, as well as extending the life of the blade. If a microtome is used that integrates the possibility of retraction, the front part of the block must be aligned with the cutting edge while the block is in descending stroke (in the forward position, not retracted)
TRIMMING OF TISSUE BLOCK
Before starting trimming, always make sure that all locking mechanisms are secure. The trimming of a block aims to completely expose the paraffin-embedded tissue in order to obtain a representative section while preserving the morphological characteristics.
MICROTOME SECTIONING: FACTORS THAT AFFECT THE THICKNESS
The microtome can be set freely, but keep in mind that there are a number of factors that determine the actual section thickness.
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The thickness of a section can be conditioned by various factors, such as the rotation speed, the angle of inclination of the blade set and the state of the blade
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The temperature of the block is one of the most important factors affecting the thickness and quality of a section
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Even the type of tissue block (soft or hard) affects the thickness especially during trimming
MICROTOMY TROUBLESHOOTING
Use a different part of the blade than the one used for grossing and If the front of the block becomes warm, it may be necessary to further cool the block.
Generally the best results with minimum compression are obtained with a slow and uniform cut. Do not interrupt and restart a cutting process, because bands of different thickness will form along the section
Before cutting each section, in some laboratories it is common to lightly blow on the front of the cooled block.
If a block is difficult to cut because it is hard or crumbly, the surface can be immersed in a bath of cold water or an emollient agent, such as a softener or a mild detergent.
For calcium-based samples it is possible to obtain sections by applying a descaling agent to the exposed tissue for 10 minutes or more. If the consistency of the sample strongly compromises the cut, especially when the sample is too soft, re-processing of the sample is necessary