In biological assays, snap-frozen OCT compounds do not retain antigenic epitopes. However, new techniques are emerging to overcome these limitations. One such technique is the use of a frozen tissue microarray. In this method, frozen cores of tissue samples are pinned into tiny holes on an Agarose-OCT block. This process preserves biological activity and freshness of the tissue microarrays.
Cryo-Gel
The present study explored the feasibility of using cryo-gel as an alternative embedding medium for OCT. This polymeric gel is highly viscous, biodegradable, and completely water-soluble. It is frequently used in biotechnology and tissue engineering. This study investigated the feasibility of using cryo-gel for snap-freezing renal biopsies and compared it to OCT-embedded samples. The Cryo-Gel-embedded tissue samples were compared with normal tissue samples, and the results showed that it preserves protein well.
The OCT and Cryo-Gel-embedded samples were compared for protein identification. The RIN values of samples embedded in the compound and those without were similar. Both methods preserved the biological activity and freshness of samples. Further, the results showed a similar pattern of protein identification in the two groups of tissue samples.
Tissue-Tek(r) OCT compound can be purchased as a single bottle of 125ml or in bulk packs of 12x125ml. Both are available with next-day delivery. Once purchased, the compound can be used to freeze tissue sections.
The process of removing OCT from OCT compound tissue samples is critical for MS analysis. Failure to remove the OCT may interfere with data normalization. The procedure involves several steps. First, the tissue sections are washed. Next, they are weighed twice. The final weight should be compared with the weight of the original tube. The difference between the two weights represents the amount of OCT compound present in the samples.
Snap-freezing
Snap-freezing of OCT compound tissue is a common procedure to extract proteins from renal biopsies. However, OCT-embedded tissue must be cleaned and de-embedded before proteomic analysis. There are numerous ways to do so, including using a solvent such as diethyl ether. In this article, we will discuss two effective methods. The first is to use an extraction agent such as diethyl ether to dissolve excess OCT in tissue.
The other method is to use liquid nitrogen or isopentane bath to prepare frozen tissue. The tissues are then laid out on the cryomold, with the correct orientation for cutting. The OCT solution is poured over the tissue carefully. The mold is then placed on an aluminum plate, which will speed up the freezing process. Once the frozen tissue is cooled, the sample should be placed into pre-labeled vials containing 1.8 mL of 10% buffered formalin, labeled with the information on the tissue.
The other method is referred to as “capsule-freezing,” which combines the advantages of OCT specimen preservation with cryovial specimen storage. The OCT compound is stored in a VCap pharmaceutical capsule or 1.5-mL cryovial. The sample is then harvested by taking a slice out of the capsule. The specimen is then sealed with OCT and embedded in an OCT block before being frozen. This method offers excellent morphological preservation while being very convenient to store.
Snap-freezing of OCT compound tissue can be advantageous for preparing renal biopsies. The OCT compound is useful in many research applications, including histomorphology, immunofluorescence, RNA/DNA, and protein analysis.
Cryo-Separation
Cryo-Separation of compound tissue is an important step in the OCT imaging process. It helps to retain maximal anatomical context. This information is critical for the spatial analysis of tissues. In addition, tissues should be oriented correctly before freezing. The tissues should be stored in prelabelled vials of 10% buffered formalin to avoid damage.
OCT-embedded samples have a number of advantages. In contrast to formalin-embedded tissue, OCT-embedded specimens do not undergo fixatives. This means that OCT-embedded tissue may represent a more accurate representation of the original tissue proteome.
In addition, OCT-embedded samples have the same morphology as tissue that does not contain the compound. The DNA and RNA yields were comparable between tissue samples embedded in OCT and tissue samples that did not. RNA yields from spleen samples were lower than those from other tissue types.
The results were encouraging. One sample of normal tissue obtained just two hours after surgery was snap-frozen using Cryo-Gel and OCT compounds, while the other two samples were embedded with neither. The samples were also embedded without the compound and were assessed for the level of background staining. The samples were then stained with H&E.
In this method, samples remain in solution at a temperature of -20o C for several months. Tissue samples may be left in the solution for months, even if the specimens are not frozen. However, it is important to note that samples that remain in solution may not freeze due to RNAlater. Once frozen, specimens should be transferred to cryovial in sterile conditions or into a mechanical -80o C freezer. For example, specimens that are intended for formalin fixation should be processed after other fresh tissue procedures, including flash freezing and embedding in the OCT compound. After this, they should be submerged in an RNA-stabilizing reagent.
Storage
There are a few simple steps to tissue freeze storage with OCT compound. Firstly, tissues are stored at -80degC and thawed at -20degC 30 minutes prior to cutting. After this, fixation is necessary using a gradient sucrose solution. Once fixation is complete, the OCT compound should be dropped on the sample tray and placed face-up in the cryostat chamber. Freezing the sample for 20 minutes is essential to prevent air bubbles from impairing the section quality.
Another alternative to liquid nitrogen is to use a dry ice solution instead of liquid nitrogen. It is important to note that the use of dry ice will have unwanted effects on OCT imaging. In addition, dry ice will leave crystals on the tissue, which may hinder the tissue’s attachment to frozen embedding media.
The OCT compound is a mixture of polyethylene glycol and polyvinyl alcohol. It is widely used for the preparation of frozen tissue sections. This compound provides a stable and convenient specimen matrix that supports the tissue, helps to reduce background staining, and prevents microtome knives from becoming dull. It is also a viable option for the storage of frozen tissue sections in a room-temperature environment. Its shelf life is 24 months.
The tissue morphology of H&E-stained frozen sections of OCT and non-OCT-embedded tissue samples was similar. However, OCT-embedded tissue samples displayed greater eosinophilic cytoplasm than non-OCT tissue samples. The samples containing OCT were more likely to have darker nuclei compared to non-OCT-embedded tissue samples.
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Interference with LC-ESI-MS/MS workflow
OCT compound interferes with the LC-ESI-MS/MS sphingolipid quantification workflow in several ways. The polyvinyl alcohol content of the OCT compound causes ion suppression and loss of signal. Furthermore, it is carried over through the single-phase Bligh-Dyer extraction step, which dramatically affects the quantification of sphingolipids.
The OCT compound interferes with standard protein and nucleic acid concentration estimation methods. This means that sample analyses based on these methods may be unreliable because large amounts of OCT compound will interfere with them. However, the results of this study will aid other scientists by providing a reproducible protocol.
In LC-ESI-MS/MS analysis, it is essential to remove OCT compound from OCT compound tissue sections. Failure to do so can affect data normalization. This is achieved by washing the section twice and weighing it twice. The final weight of the tissue section is then compared to the weight of the tube in which it was frozen. The difference between the two weights represents the concentration of the Othe CT compound.
Method for preserving mucins and glycolipids
When it comes to processing OCT compound tissue for MS analysis, removing OCT is an essential step, as it may interfere with the normalization of data. There are several steps involved in removing OCT. First, the sections must be washed. After washing, the tissue sections should be weighed twice. The difference between the two weights should be compared to calculate the OCT content. Typically, this difference is between 14.7 mg and 375.7 mg.
The method for preserving mucins and glycolipolipids in OCT compound tissue preserves the natural distribution of these molecules, which are important for microbial-host interactions. Furthermore, understanding the distribution of mucins and glycolipids can provide new insights into host defenses and pathogens.
Although mucins are known to play an important role in disease diagnosis and prognosis, a number of challenges remain. These include the need for new strategies for the production and characterization of mucin-like molecules. Furthermore, a deeper understanding of the role of mucins in disease progression is needed to develop novel vaccines and therapeutic agents.
The glycocalyx layer is a highly hydrated glycoprotein-rich coating that shrouds many eukaryotic cells. The intestinal epithelial glycocalyx is made of glycosylated transmembrane mucins and is essential for nutrient absorption. Many gastrointestinal diseases result from the disruption of the intestinal epithelial glycocalyx.
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