OCT Compound Tissue Freezing can be accomplished in a few simple steps. Generally, tissues are stored at -80degC and should be thawed at -20degC 30 minutes before use. The fixation of tissue is usually done with gradient sucrose solution. After the fixation is complete, drop the OCT compound on the sample tray, and face up. The tissue should then be placed in a cryostat chamber or quick-refrigerating shelf. The tissue should be frozen for 20 minutes in order to prevent air bubbles from affecting the quality of sections.
Staining with ColorBond(tm) mordant
OCT Compound is a water-soluble blend of resins and glycols that provides a convenient specimen matrix for cryostat sectioning. It also prevents background staining, leaving no residue on slides. It also prevents microtome knives from dulling and allows frozen tissue sections to be stored at -20 to -80°C.
When choosing a staining method, the individual pathologist should consider the advantages and limitations of each. Choosing the best staining method will be based on your personal training and experience, but it is important to note that some methods have more advantages and disadvantages than others. Different techniques and brands of solutions have distinct characteristics, and understanding the principles behind these differences can help you troubleshoot any issues. It is critical that staining is reproducible and accurate in order to improve the quality of pathologic diagnosis.
The mucus layer on unfixed frozen tissue is rough and may detach from the tissue. The thickness of this layer is similar to that of paraffin-embedded tissues and Carnoy’s solution-fixed tissues. In fact, the mucus layer thickness of a frozen human colon tissue section is approximately 100 mm.
Many laboratories have tissue samples frozen in OCT and use these frozen tissues to study mucus-associated glycans and glycolipids. This minimally processed approach preserves the natural distribution of mucins and glycolipids, which are important for microbial-host interactions. Knowing how these molecules are distributed in the tissue can lead to a better understanding of pathogens, host defense, and microbial exploitation.
Removal of OCT from frozen sections
In order to remove OCT from frozen OCT compound tissue sections, you must first remove the OCT compound itself. The compound interferes with multiple elements of the LC-ESI-MS/MS workflow. Specifically, OCT interferes with lipid extraction, LC column stability, and MS/MS instrument contamination.
Frozen tissue sections must be prepared in sterile conditions and preferably at a low temperature. After freezing, tissue sections should be cut into small pieces. If multiple sections are required, the tissue should be prepared in several layers. This minimizes the possibility of error and helps in the identification of primary pathology. Once cut, the tissue should be placed on an antiroll plate. After sectioning, the frozen section should be immersed in methanol (95 percent) for one minute. The fixation process must be completed quickly to prevent the appearance of swollen cells and hazy cell margins.
Removing OCT from frozen OCT compound tissue sections is an important step for MS analysis. However, it can interfere with data normalization. Because of this, the removal process involves several steps. First, it is essential to wash the sections. Then, they must be weighed. The weights of tissues should be weighed twice before removal. The final weight of the tissue sections should be compared with the initial tube weight. The difference between the two weights is the OCT compound content, which varies between 14.7 mg and 375.7 mg.
Once the frozen OCT compound tissue sections are thawed, they are prepared for further analysis. The tissues are then centrifuged at 5,000 g for 10 min. The lipids in the tissues are analyzed using LC-ESI-MS/MS.
The process of removing OCT from frozen OCT compound tissue sections is similar to the procedure of preparing frozen tissue sections. Once the tissues are thawed, they are placed in a 15 ml polypropylene conical tube containing 10 ml of prechilled ice-cold deionized water. Following this step, the tissues were maintained at 4degC, and then the steps for sOCTrP were performed on human tissues.
This study found that tissue morphology was similar in both OCT-embedded and non-embedded tissue sections. In addition, the RNA yield and DNA quality were comparable. However, spleen tissue samples had lower RIN values than other tissues.
To remove OCT from frozen OCT compound tissue sections, Cryo-Gel was used. Then, samples were processed with a digest buffer containing 0.1% Rapigest and 50 mM ammonium bicarbonate. After the samples were digested, protein samples were lysed with 70% amplitude sonication. After this, the samples were incubated for 30 minutes in an alkylation solution containing 300 mM iodoacetamide. The samples were then evaluated using multiplex PCR.
Issues with snap-frozen tissues
Snap-frozen OCT compound tissues are used in intraoperative pathology procedures. These tissues are placed on a chuck metal with optimal cutting temperature for a rapid freeze. Although these tissues are often soft, the freezing process can result in ice crystals. Fortunately, liquid nitrogen is available to fix this issue. The drawback to using liquid nitrogen is that it rapidly evaporates, meaning the container must be refilled several times a day.
When using snap-frozen OCT compound tissues, it is important to use the appropriate rehydration solution for each tissue type. Once a tissue block is frozen, it can be stored at -80deg C for up to six months. After that, it can be restored to room temperature.
Snap-frozen OCT compound tissues can be stored in plastic bags. These can then be grouped with other samples in a cryo-container. However, as the cryo-container fills with samples, it can become unmanageable and difficult to locate.
In addition to being fragile, snap-frozen OCT compounds are not very stable. Moreover, they do not retain the antigenic epitopes. This can cause a variety of problems when performing biological assays. However, there are new methods to solve these problems. For example, a new method is used for preparing frozen tissue microarrays. The frozen cores of these samples are pinned into tiny holes on a block made of Agarose-OCT. The freeze-thawing process also preserves the natural biological activity and freshness of these tissue microarrays.
Another method for snap-frozen OCT compounds is using a dry ice mixture instead of liquid nitrogen. This method can also be used for tissue freezing in the OCT compound. A solution of isopentane/2-methyl butane is used to snap-freeze tissue, but the use of dry ice may have undesirable effects on OCT imaging. In addition, the presence of ice crystals on the surface of the tissue makes it difficult to attach to frozen embedding media.
Snap-frozen OCT compound tissues are not suitable for all purposes. The processing of these tissues must be done safely and properly. It is important to check the surgical schedule and notify the pathologist on call before completing the process. Depending on the type of tissues used, the fixation temperature may need to be adjusted.
Snap-frozen OCT compound tissues are also susceptible to adherence problems. Tissues can be difficult to embed on slides when they are fixed with formaldehyde. Alternative fixation methods, such as 15% sucrose in 1xPBS, may yield better results.
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