Freezing tissue liquid used

Fig. 4.2 Method of freezing tissue. To freeze tissue rapidly, the amount of material frozen needs to be as small as possible. Freezing tissue with no liquid is best. On a strip of aluminum foil, write the sample information, place a blotted price of tissue on the foil, and plunge in isopentane. Freezing tissue surrounded by liquid has a slower rate of freezing, but this method is sometimes used. Place a piece of tissue in a mold, cover with O.C.T., and plunge in isopentane...

O.C.T. (optimum cutting temperature) - is an embedding liquid used to surround tissue blocks for freezing on a cryostat chuck. 

Snap freezing - widely used term to describe freezing tissue for immunocytochemistry the term comes from food preparation industry and for biomedical sciences it has no single definition and can mean freezing in liquid nitrogen, on dry ice, or in isopentane. 

Freeze tissue in liquid nitrogen in a mortar, grind to a powder with a pestle. Cool the mortar and pestle prior to use with liquid nitrogen, and work quickly so as to avoid thawing of the frozen tissue. 

Dispose of the solution containing formaldehyde and glycine, rinse the seedlings three times with Milli-Q (deionized) water, remove as much water as possible using tissue paper, transfer the plant material to a new 50 mL tube, and flash freeze in liquid nitrogen (see Note 6). 

Freeze tissue directly in liquid isopentane after dissection, cool on dry ice, and store at —80°C until use. Postmortem changes are known to occur in the proteome of susceptible peptides and proteins within minutes. To prevent these alterations of the sample, the dissected tissue must be snap-frozen and defrosting of the sample should be done only just before sample preparation starts (rrr Note 1). 

Skin contact Contact with liquid nitrous oxide can freeze tissue. In case of frostbite, place the frost bitten part in warm water, 100°F to 105 F (37.8°C to 40.6°C). If warm water is not available or it is impractical to use, wrap the affected part gently in blankets. Do not rub. Consult a physician. 

Liquid nitrogen (subcooled) -210°C Only useful for freezing well-cryopro-protected specimens, e.g., sucrose-infused tissue... 

The ideal solution to microanalysis would be simply to freeze the plant material rapidly to the temperature of liquid nitrogen and then section it while it is still frozen on a cryotome. The frozen sections would then be transferred to a cold stage in a TEM and analyzed. In theory, no ion movement will take place and analysis at the high resolution of TEM should be possible. Indeed, this is a useful technique for liver, kidney, and soft animal tissues, but unfortunately it is almost impossible to cut tough plant material, and maintain the sections in a reasonable state for analysis (2). Even if this problem could be overcome unstained tissues will be difficult to visualize in TEM. 

Rapid freezing is the best preservation tool. The use of liquid nitrogen and cooled isopentane are for the purposes of cooling quickly. The tissue should be relatively small in size to allow for rapid and thorough penetration of the cold-temperature chemicals. 

Before the extraction procedure may commence, the sample must be prepared in such a way that it is in a condition for extraction of the analyte(s). For analyzing sulfonamide residues in liquid samples such as milk, a pretreatment dilution step with water prior to direct fluorometric detection may be required (207). Dilution of milk with aqueous buffer (208) or sodium chloride solution (209) prior to sample cleanup has also been reported. For the analysis of honey a simple dissolution of the sample in water (210, 211) or aqueous buffer (212) is generally required. Semisolid samples such as muscle, kidney, and liver, require, however, more intensive sample pretreatment. The analyte(s) must be exposed to extracting solvents to ensure maximum extraction. The most popular approach for tissue break-up is through use of a mincing and/or homogenizing apparatus. Lyophilization (freeze-drying) of swine kidney has been carried out prior to supercritical-fluid extraction of trimethoprim residues (213). 

Kidney tissue is fixed with paraformaldehyde-lysine-periodate by vascular perfusion (Brown et al., 1996). Tissue slices are further fixed overnight at4°C with the same fixative and stored in PBS (pH 7.4) containing 0.02% sodium azide. They are placed in 30% sucrose in PBS for at least 1 hr, and then surrounded by a drop of Tissue-Tek embedding medium on a cryostat chuck before freezing by immersion in liquid nitrogen. Cryostat sections about 5 p,m thick are cut at a chamber temperature of -25°C, collected on Fisher Superfrost Plus charged slides, and stored at —20°C until use. 

In cryosurgery, liquid nitrogen is used to fast-freeze cancerous tissues and warts. This proven new technique kills the cancerous area and allows surgeons to safely remove the dead tissue. 

The sensitivity quoted above is for a dry sample and this is greatly reduced by the presence of water. This raises difficulties in the study of samples such as tissue slices, blood, or cell cultures in in vivo conditions. Specially shaped sample holders have to be used and the sensitivity is reduced by a factor of about 100 (allowing for the smaller volume of sample which can be introduced into the spectrometer). A way round this difficulty is to freeze the sample (which restores the full sensitivity), and then to record the spectrum at a low temperature, usually that of liquid nitrogen (77°K). 

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