OCT Compound Tissue Freezing is a technique that uses a compound that freezes tissues to reduce artifacts and ensure tissue integrity. The compound is commonly known as isopentane-2-methyl butane and it is essential to apply the solvent to all areas of the tissue and allow it to settle for at least 30 seconds.
RNA analysis
In the present study, we evaluated the robustness of OCT by evaluating the RIN values of H&E-stained tissue sections from two tissue types: muscle and liver. These samples were subjected to three different freezing protocols. The results were similar in terms of morphological integrity, RNA purity, RIN values, and performance. The minimal values for evaluable areas of samples directly frozen in liquid nitrogen met our acceptance criteria, and no outlier values were identified.
The most robust method used in this study was the direct freezing of liver tissue with liquid nitrogen. This method exhibited a reproducible CV (CV) of 10.7% and an interspecies CV of 25. In addition, the RNA quality was comparable between the three different tissue types. However, OCT had a negative impact on liver tissue samples, as demonstrated by the lower RIN values. Despite this, we found no significant differences in gene expression analyses after OCT treatment.
After freezing the samples, the RNA was extracted using RT-qPCR. The analysis of the samples showed comparable expression levels of Alb and Apoh in the muscle and liver tissue. Likewise, Ckm expression levels were comparable. Therefore, all three sample types were equally suitable for RNA analysis downstream.
In addition to OCT compound tissue freezing, OCT is also an effective method for RNA analysis. It provides a stable matrix for frozen tissue specimens and eliminates background staining. The specimens are packaged individually in high-density polypropylene cryotubes with a small rubber o-ring for sealing. The samples are then placed in liquid nitrogen tanks.
OCT compound tissue freezing has been used widely in the surgical suite for the preservation of tumor tissue for histopathologic analysis. However, OCT compounds pose a challenge to LC-MS/MS experiments. Furthermore, protein recovery from OCT-embedded NSCLC tissue samples requires a step of trichloroacetic acid precipitation.
However, a well-monitored freezer is capable of preserving high-quality RNA for decades. As RNA is the most rapidly degradable component in tissues, long-term preservation in -80degC freezers is a sign of an appropriate storage routine.
Preparing tissues for OCT compound tissue freezing
Preparing tissues for OCT compound tissue freeze-drying requires some care. The tissues should be thawed to -20degC about half an hour before the procedure. Once frozen, the tissues should be oriented so that the cut surface is at the bottom. Carefully pour 2 drops of OCT onto the frozen tissues. The tissue mold should be placed on a plate with dry ice, or a cryostat chamber, to expedite the freezing process. The tissue should be frozen for approximately 20 minutes to avoid air bubbles in the samples.
After the tissue blocks are frozen, they must be embedded in the OCT compound. The OCT compound is a gelatinous compound that stabilizes the tissue during cryostat sectioning. The freezing process is rapid to avoid ice crystal formation. However, there are some risks associated with frozen tissues.
To achieve high-quality images, the underlying tissues must be properly prepared. This requires careful handling and proper documentation. The process involves various steps, each of which must be performed safely. The first step is acquiring the specimens. Before the process begins, the specimens must be inspected for quality control.
To prepare tissues for OCT compound tissue freezing, a metal chuck with the appropriate cutting temperature is used. A liquid nitrogen chamber is an alternative method for freezing tissues. In addition to liquid nitrogen, supercooled liquids can also be used for the freezing process. These are used for tissues in the surgical pathology field.
Several laboratories keep tissue samples frozen for OCT compound tissue freezing. These frozen tissues are often used to study the distribution of glycolipids, mucins, and glycans. This minimally processed technique allows scientists to maintain the natural distribution of glycans, which are hydrophobic and are crucial for understanding host defense, microbial exploitation, and pathogens.
OCT compound tissue freezing is a convenient way to freeze tissue for OCT imaging. It preserves the integrity of mucus-associated glycans and glycolipids. OCT compound tissue freezing is an excellent method for examining mucus-associated glycan structures in the brain.
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Storage
Tissue freeze storage is an important step for tissue analysis. Using OCT compound is a safe and effective way to maintain tissue freezing temperatures. It is non-conductive by nature but can be converted into a conductive material by adding carbon powder or colloidal graphite. In addition to tissue freeze storage, OCT compound can also be used in cryo-SEM applications.
However, there are some problems with this method. First of all, the OCT compound may interfere with the integrity of the MS/MS instrument. Additionally, it can interfere with multiple elements of the LC-ESI-MS/MS workflow. Therefore, removing OCT from tissue samples before freeze storage is crucial.
Another important step in tissue freeze storage is dissecting the tissue block to obtain high-quality tissue sections. If the tissue is not well cut, the immunostained sections will be hard to interpret. Moreover, the resulting sections can be damaged if improperly cut. OCT compound must be re-applied to the cut surface to prevent desiccation of remaining tissue in the block. After that, the cut sections can be stored at -20deg C using a desiccant. The time of storage should be correlated with the reactivity of each antigen and antibody pair.
OCT compound tissue freeze storage should be done in sterile conditions. The tissues must be cut into small pieces and should be prepared in several layers to minimize the risk of error and improve the identification of primary pathology. These sections should then be mounted on gelatin-coated slides. During the fixation step, it is important to maintain the temperature between -15 and -23 degC so that ice crystals do not form on the slides.
Tissue samples that were stored in OCT compound and without compound were comparable in morphology. Tissue samples embedded in the OCT compound had a greater proportion of eosinophilic cytoplasm than tissue samples that were not. The samples that were not embedded in the OCT compound were less darkly stained when stained with H&E.
The proteins from OCT compound tissue can be extracted for proteomic analysis. The process requires the use of a software program called Scaffold, which estimates a 90% probability of the presence of proteins in samples.
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