If you want to prepare tissues for OCT Compound Tissue Freezing, there are some things you should do before the procedure. You should prepare tissues and storage containers for cryopreservation in the correct manner. This will ensure that your tissues are preserved in the best possible way. The solution that you should use is isopentane-2-methyl butane or a similar solvent. The solution should be applied to all the areas of tissue, and it should be left to settle for at least 30 seconds.
Issues with OCT Compound Tissue Freezing
The procedure for tissue freezing using OCT compound involves embedding a frozen block of tissue in a gelatinous mounting medium. The tissue is then immersed in a solution of 2-methyl butane and alcohol and rapidly frozen in liquid nitrogen. This procedure stabilizes the tissue during cryostat sectioning. However, it must be done rapidly because of the risk of ice crystals forming.
The process of snap-freezing OCT compound tissues is used for intraoperative pathology procedures. These tissues are placed on a metal chuck at the right temperature for rapid freezing. However, the ice crystals in the tissue are not always removed, and it is necessary to replenish liquid nitrogen frequently. The tissues also need to be rehydrated with appropriate rehydration solutions. Snap-frozen tissue blocks can be stored at -80 deg C for up to six months. Once restored to room temperature, the tissue blocks can be used again.
Another method for tissue freezing is flash-freezing. This method is more expensive and requires the preparation of a special compound. The solution used to freeze specimens is not free from impurities and is opaque near the freezing point. Moreover, the specimens can be stored at -20 to -70 degrees Celsius for several months.
A common problem with snap-frozen OCT compounds is that they do not retain the antigenic epitopes. These flaws can lead to problems in biological assays. Fortunately, new techniques are now available to overcome these problems. One such method uses an Agarose-OCT block to prepare frozen tissue microarrays. The frozen cores of the tissue samples are then pinned into tiny holes on the Agarose-OCT block. The process preserves the freshness of the tissue microarrays and preserves biological activity.
Another method for snap-freezing tissue is called optimal cutting temperature compound (OCT). These compounds contain ethylene glycol and polyvinyl alcohol. These nonreactive ingredients help stabilize the tissue and provide a smooth cutting surface. They also preserve morphological characteristics.
Preparation of tissues for OCT Compound Tissue Freezing
The first step in the preparation of tissue sections for OCT compound tissue freezing is to freeze the tissue block. The tissue block is then embedded in a gelatinous OCT compound. This solution is then quickly frozen in liquid nitrogen. This step is essential for preserving the tissue during cryostat sectioning. The freezing procedure must be done as quickly as possible to avoid ice crystal formation.
To prepare tissue samples for OCT compound tissue freezing, the tissue must be frozen at -80degC at least 30 minutes before freezing. After thawing, the tissue must be fixed and dehydrated using a gradient sucrose solution. Once fixed, the tissue sample should be placed on a cryomold and covered with the OCT compound. The mold should be placed on an aluminum plate on dry ice to speed up the freezing process. The sample holder should then be placed in the cryostat chamber or quick refrigerating shelf and frozen for at least 20 minutes. This step is essential to avoid air bubbles in the specimen, which can affect the quality of the section.
After freezing, the tissue is embedded in a gel or glue. The process is similar to that of unfrozen tissue. The frozen tissue must be frozen as quickly as possible to avoid ice crystals and bad morphology. OCT compound is applied to frozen tissue using a Peel-A-Way freezing mold. The tissue block can be sectioned from the bottom or sides.
When the tissues are ready for OCT compound tissue freezing, the pathologist must prepare them for use. It is important to protect the tissues from drying by wrapping them in plastic centrifuge tubes and foil envelopes. It is also a good idea to double wrap tissues to keep them from wilting.
Storage of tissues in a cryo-container
OCT compound tissues are best stored in a cryo-container until they are needed. Before storing them, they need to be properly labeled. The label should be in permanent marker and should fit the size of the sample. Once labeled, the OCT compound must be placed in the center of the bottom of the cryomold. The tissue sample should be oriented so that the side touching the bottom of the cryo-mold is the side that you plan to section first.
OCT compound tissues should be stored in a cryo-container that contains a block to prevent desiccation. Moisture should also be added to the block before freezing to prevent the specimen from dehydrating in a cryo-container. This will also ensure the sample is not difficult to section because of the water content.
The OCT compound should be stored at -20degC to prevent desiccation and preserve its integrity. However, it is important to thaw tissue to -20degC at least 30 minutes before use. After dehydration, the tissue should be reapplied with OCT to the surface of the cut tissue to protect the rest of the block from desiccation. After the procedure, the tissues can be stored in a cryo-container at -20degC in the presence of a desiccant. Ensure that the tissue does not have air bubbles in it and is not too thin.
OCT compound tissues should be stored in a cryo-container containing 1.8 ml of 10% buffered formalin. A copy of the consent form must be kept with the tissues. If the tissue is a frozen section, the pathologist on call should be notified.
Dry ice for OCT Compound Tissue Freezing
Tissue freezing is one of the most important steps in OCT compound tissue preparation. The tissue must be frozen immediately and kept at -70 deg. Then, it should be embedded in the OCT compound. This compound is a gelatinous solution that is used to preserve the tissue specimen during cryostat sectioning. The tissue must be frozen quickly to avoid ice crystal formation. After the tissue has been frozen, the sections should be air dried.
For OCT compound tissue freezing, dry ice pellets can be used. To prepare the cryomold, a small stainless steel bowl can be placed in a styrofoam container. This bowl can then be filled with isopentane or acetone. Once the mixture is ready, the tissue can be placed in a pre-labeled vial.
Dry ice is an ideal freezing medium for OCT compound tissue preservation. It freezes tissues in a fraction of the time when compared to freezing tissues in a freezing medium. In addition, it is safe and easy to use. The only drawback is that the tissue does not freeze as fast as it would if it were immersed in the liquid.
Dry ice for OCT compound tissue freezing is a useful technique that allows tissue to be kept at -20o C for several months. However, it is essential to keep the tissue out of the freezing solution as much as possible. This can help prevent the formation of ice crystals and prevent the specimen from being damaged. It is also important to keep handling the frozen specimens to a minimum to prevent thawing.
Technique for sectioning OCT Compound Tissue
When preparing OCT compound tissue sections, it is important to adhere to a few basic procedures. First, the block must be frozen. This will ensure that the tissue will not dehydrate and is not prone to desiccation, which can cause difficulty in sectioning. Second, the tissue should be moistened before it is embedded in the block. This will prevent it from becoming too fatty or watery, and it will also prevent ice crystals from forming inside the section.
The OCT Compound is a blend of clear water-soluble glycols and resins. It provides a solid matrix for tissue specimens and eliminates background staining. It is also fast-freezing and available in squeeze bottles. Typically, one bottle contains enough material to make 12 frozen section blocks.
After embedding the tissue in the OCT compound, it should be immediately frozen in isopentane (or dry ice). The tissue block should be frozen at -70 deg. Afterward, the sections should be sectioned at 5-10 um and mounted onto gelatin-coated slides. It is important to keep the cryostat temperature between -15 and -23 deg. If the tissue is too cold, it will curl. Then, the tissue block should be air-dried to prevent the sections from falling off the slides.
Ideally, the tissue is positioned perpendicular to the blade. This is referred to as the critical edge. The tissue is divided into three zones: the beginning, the middle, and the end. The beginning and end will often curl or stretch due to the initial hesitation in engaging the tissue. In contrast, the middle zone is the smoothest path with clean histology.
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