Freezing tissue

Think about freezing methods in terms of the tissue samples and the desired outcome. Initially, determine the size of the tissue block, the number of tissue blocks to be sectioned at one time, and the requirements for specific orientation of the tissue. Because most scientists need a particular orientation of their section, the tissue must be frozen in a specific position to give the needed sections. 

Once infiltrated with sucrose, the tissue block is frozen by surrounding it in a liquid, which holds the frozen tissue for sectioning. The most common liquid is optimum cutting temperature or O.C.T. (Sakura Tissue-Tek) another embedding liquid is tissue freezing medium or TFM (Triangle Biomedical Sciences). 

Isopentane (Sigma M32631) is the best freezing agent for sucrose-infiltrated tissue for immunocytochemistry. In a plastic beaker that is cooled in liquid nitrogen, add the liquid isopentane (Fig. 4.2). If liquid nitrogen is not available, a dry ice alcohol slurry works, but it is not as cold. Isopentane will freeze solid white in liquid nitrogen, so to thaw frozen isopentane push a piece of metal, such as the blade of a screw driver, into the isopentane until liquid. 

A preferred method for freezing samples is to use strips of aluminum foil (7- to 8-mm wide and 40-mm long) with an identification pressed in at one end with a pencil and the sample sitting on the other. Remove the tissue from the 20% sucrose solution, blot on a piece of paper towel, and set on the other end of the foil. Immediately with a forceps, plunge the foil strips with the tissue into the cold isopentane (Fig. 4.2). The tissue will stick to the foil and it can be placed in a vial in the liquid nitrogen bath and is subsequently stored in a -70°C freezer in individual small tubes. For sectioning, the frozen tissue is mounted later in O.C.T. on a chuck. 

Another method for freezing samples uses plastic molds. There are many varieties of small disposable plastic molds for one or more pieces of tissue to be positioned next to one another. Rather than freezing the tissue blocks individually 

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Liquids functioning through physical action applied to the body to cool or freeze tissues for therapeutic purposes. 

Smolenska-Sym, G., and Kacperska, A., 1996, Inositol 1,4,5-trisphosphate formation in leaves of water oilseed rape plants in response to freezing, tissue water potential and abscisic acid. Physiol. Plant 96 692-698. 

If no other histochemical techniques are needed, fresh tissue can be used. The block must be frozen quickly without cracking. Tissue is cut in small blocks with a new razor blade (washed in soap to remove oil). An alternative to the method below is freezing tissue on aluminum foil on top of dry ice and surrounding with crushed dry ice. However, the perimeter of the tissue is usually poorly preserved with this method. For RNA detection, DEPC is included at 0.02% in the subbing and fixation solutions. Poly-L-lysine coating may be superior at temperatures > 50°C since gel from gel-coated slides may detach at higher hybridization temperatures. It is convenient to use only some of the slides for immediate hybridization and store the rest for future reference. 

Place a beaker in liquid N2 and fill with Cryokwik, isopentane or Freon. These coolants freeze tissues much faster than N2 and prevent the formation of large ice crystals that would damage the cellular structures (Terracio and Schwabe, 1981). Stirring of the coolant can be beneficial as it increases the cooling rate. 

Following infiltration with sucrose, the next step is freezing the tissue so that it will be held solid during the sectioning. There are a wide range of methods used for freezing tissues we will look at the strengths and weaknesses of several of these methods. 

There are four basic ways of freezing tissue or cells (Fig. 4.1). The most rapid freezing, vitrification, which holds each water molecule in place without the presents of ice crystals (Fig. 4.1, pathway No. 1). This method is difficult to perform and involves slamming the tissue onto a silver block at liquid helium (-214 C) temperature. For light microscopic immunocytochemistry, this method is not practical. 

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...

Paraformaldehyde in phosphate buffer fix 20% Sucrose in PBS Styrofoam and beaker for freezing tissue Aluminum foil... 

A second approach to postembedding electron microscopic immunocytochemistry is the use of special aqueous resins, while these resins are still hydrophobic, they tolerate some aqueous material in the tissue. Examples of these resins are LR White or Lowicryl K4M. Another approach with aqueous embedding is to freeze tissue in buffer very rapidly, and to section with a cryo-ultramicrotome. Cryo-ultramicrotomy is a technique requiring considerable training and cannot be used by the novice. 

Isopentane - a chemical used for rapid freezing tissue blocks at -160°C. 

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. 

X 1.5 cm to allow for quick freezing. Tissues may remain in isopentane for long periods without harm. A minimum of 2-3 min should be allowed for complete freezing. Specimens are then transferred to a container with dry ice for transport to the biorepository. 

Five minutes immersion is sufficient to freeze tissue. Check for firmness by grasping with gloved fingers or probing with tweezers. If it feels solid, it is frozen. 

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. 

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). 

After snap freezing, tissue is removed from isopentane and stored at -80 C. We heartily recommend not surpassing a storage period of 6 months. After 6 months of storage, variations in the molecular profiles are observed if no sample stabilization procedure is performed. Preferentially, tissues should be analyzed a few days or weeks after snap fteezing. 

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. 

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