OCT Compound Tissue Freezing (OCT) is a compound used in the tissue-freezing procedure. OCT is a compound that is poured into a plastic cryomold and poured over the tissue. The mold should be placed on an aluminum plate containing dry ice to accelerate the freezing process. When the freezing process is completed, the frozen tissue should be placed into a pre-labeled vial containing 1.8 mL of 10% buffered formalin.
Cryo-Gel
One of the main objectives of renal biopsies is to preserve the tissue for later diagnostic studies. To that end, tissue samples are often snap-frozen using Cryo-Gel as the embedding medium. In this study, we compared the use of OCT with that of Cryo-Gel.
The Cryo-Gel for OPT Compound is designed to be both protective and supportive. The freeze-dried tissue sections are used for rapid examination in the laboratory. The freezing process is quick and efficient, and the gel stabilizes the tissue during cryostat sectioning.
Similarly, OCT and Cryo-Gel samples were similar for protein identification. However, the OCT sample showed repeating polymer peaks, which interfere with the MS signal. This polymer is not a problem with Cryo-Gel.
To use Cryo-Gel for OPT Compound Tissue Freezing, it is best to prepare the tissue and fix it beforehand. Then, you should cool the sample with isopentane using a liquid nitrogen can. After cooling, place the specimen in an OCT-filled cryomold, preferably on dry ice. The tissue should be preserved at -80degC for up to 12 months.
Cryo-Gel for OCT Compound Tissue Freezing is an ideal mounting medium for frozen tissue specimens. This compound contains a blend of non-reactive ingredients, which provides a solid matrix around the tissue specimen. It also eliminates background staining and makes for fast freezing. The compound is available as a squeeze bottle or as a 12-pack.
OCT Compound is a water-soluble blend of glycols and resins, which provide an excellent specimen matrix for cryostat sectioning. Additionally, unlike other types of compound tissue freezing, it won’t leave any residue on slides, which eliminates unwanted background staining. In addition, OCT Compound is non-abrasive, meaning that it will not cause microtome knives to dull.
OCT
OCT compound tissue freezing is an excellent way to preserve tissue samples during IHC. The process begins with a block of frozen tissue, which is then embedded in a mounting medium. This mounting medium is a gel-like compound made of OCT. This gel-like compound is then immersed in a liquid nitrogen solution and quickly frozen. The gel-like OCT compound helps stabilize the tissue and prevents it from breaking apart when frozen. This method prevents ice crystals from forming, which would cause a significant loss of reactivity.
No compound
The study examined tissue morphology in OCT-embedded liver and renal tissue samples. OCT-embedded samples showed more eosinophilic cytoplasm, and H&E-stained samples showed darker nuclei. The researchers concluded that OCT is a promising technique for assessing tissue morphology in the kidney and liver.
OCT is a water-soluble compound made of polyethylene glycol and polyvinyl alcohol that has been widely used in the preparation of frozen tissue sections. It improves tissue continuity and minimizes the chipping, wrinkling, and pleating of the sample. It can also minimize background staining. OCT frozen sections can be stored for up to 24 months.
The procedure for OCT tissue freezing is similar to that of unfrozen tissue freezing. The first step is to freeze the samples. The second step involves dehydrating the tissue with a gradient-sucrose solution. The third step involves placing the sample holders into a liquid nitrogen storage tank or a quick refrigerating shelf. Once frozen, the samples should be labeled with the sample ID.
OCT tissue freezing can be used to isolate proteins in tissues. For example, protein extraction from OCT-embedded human kidney tissue can be performed using LC-MS/MS proteomics. The study authors published imaging mass spectrometry protocols in 2010 and 2014. The authors of this study have also published their protocols and suggested the optimal cutting temperature to remove the compound.
The compound used in snap-freezing biopsy material is called optimal cutting temperature compound (OCT). The compound contains polyethylene glycol, polyvinyl alcohol, and other nonreactive ingredients to stabilize the tissue. These substances also ensure smooth cutting surfaces and preserve the morphology of the tissue.
RNAlater
RNAlater is a proprietary fixative designed to preserve the integrity of RNA in tissue samples. It can be used instead of traditional freezing methods to preserve sections of tissue. The procedure can preserve tissue morphology and architecture without affecting sample staining. It also allows for long-term storage of tissue samples in liquid nitrogen.
RNA is stable for a number of hours in nonfixed tonsil tissue, and after overnight storage in RNAlater, the RNA quality remained unaffected. The same conditions were applied to fresh colon tissue, with a similar result. In addition, RNAlater preserves RNA by allowing the sectioning of frozen tissue without degradation.
To ensure optimal results, specimens should be stored in RNAlater overnight at a temperature of 2o-8o C. However, they can be stored at -80o C for up to 2 weeks. In addition to using RNAlater, specimens should be transferred to cryovials or mechanical -80oC freezers. These fresh tissue processing procedures should be performed after other fresh tissue procedures such as flash freezing and embedding in OCT compound. The final step in the process should be submersion in an RNA-stabilizing reagent.
In addition to ensuring high-quality RNA, RNAlater has other applications. For example, it can be used to treat tissue cryosections, develop staining protocols, and prepare tissues for laser microdissection. These techniques require high-quality, RNA-rich tissue samples.
RNAlater has also been used to isolate RNA from tissue sections. The preparation of tissue sections with RNAlater yielded a high yield of RNA, with a remarkably high purity of RNA. It was also shown that snap-frozen mouse kidney tissue yielded RNA with an A260/280 ratio of 1.91.
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Snap-frozen tissues
Snap-frozen OCT compound tissues are often used to preserve small pieces of frozen tissue. These samples are fixed using a fixative (paraformaldehyde or neutral-buffered formalin) to inhibit the growth of microbes and to improve their morphological stability. Snap-frozen OCT compound tissues can be difficult to retrieve and cut into sections, particularly when they are small and irregular.
In the present study, two samples of normal renal cortex and two samples of surgical resections were snap-frozen using different embedding compounds. One sample was embedded in Cryo-Gel and one was in Tissue-Tek OCT compound. Using both embedding compounds, the tissues were analyzed by a pathologist using a Leica CM 1950 cryostat at -20 degC, within a week of snap-freezing. The tissue samples were then mounted on the cryostat microtome object holder using a small drop of NaCl.
OCT-embedded human kidney tissue is suitable for LC-MS/MS proteomics. However, some disadvantages of OCT-embedded tissues include the presence of water-soluble synthetic polymers in the sample, which may interfere with ion formation. Additionally, the polymer peaks in OCT may hide smaller protein peaks. Finally, removing OCT from samples is a complicated process, which can lower the protein yield.
In this study, we investigated whether the morphology of OCT-embedded tissues remained similar to that of those without the embedding compound. We also found that DNA quality was similar in both methods and that the RNA yields were similar. In contrast, the RNA yield was lower in the spleen sample than in the other tissues.
Moreover, the new method involves only one freeze-thaw cycle before biological assay. This minimizes the damage to cellular architecture, antigenic epitopes, and nucleic acid structures, as well as the enzyme and receptor activities. In contrast, previous methods required several cycles of freezing and thawing cell or tissue samples.
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