Cryoprotected tissues

Fig. 4.8 Freezing microtome. To cut thick frozen sections of 15-50 (rm, a freezing microtome will do the job. The cryoprotected tissue is frozen in O.C.T. on the chuck with hquid CO2, a knife is brought across the tissue, and the section is collected with a small paint brush and placed in a tray...

Place the fixed, cryoprotected tissues into a mold (fashioned from aluminum foil), containing OCT compound... 

Finkle BJ, ZavolaME, Ulrich JM. Cryoprotective compounds in the viable freezing of plant tissues, in Cryopreservation of Plant Cells and Organs (Kartha KK, ed.), CRC Press, Boca Raton, FL, 1985, pp. 75-113. 

For cryostat sectioning, the tissue specimens are cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 12 hr or until they sink to the bottom of the container. They are embedded in O.C.T compound (Miles, Elkhart, IN) and frozen in N-heptane cooled to the temperature of liquid nitrogen. Alternatively, if the antigens are resistant to paraffin embedding, the specimens can be dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. 

Recently, it was reported that a freshly-taken, fully hydrated full thickness human epidermis can be completely vitrified, directly, without the use of cryoprotectants or any other pretreatment (Norlen et al., 2003). As mentioned above, successful tissue vitrification has the potential to preserve biostructures down to atomic resolution. Consequently, the native ultrastructure of epidermal biomolecular complexes could now therefore, theoretically, be observed at subnanometer resolution in situ. 

The matter of cryoprotection, with clinical applications in mind, will undoubtedly become better understood with time. Obviously, measures of cell or tissue viability must be thought of with more subtle and stringent criteria in mind than those normally used in most studies of tissue preservation. Assessing viability in terms of one or a few bio-... 

It is not known whether such natural cryoprotectants occur widely distributed among animal species. However, a relevant experiment was conducted with brain slices, from warm-adapted and hibernating hamsters, before and after freezing (92). Tissue slices from the hibernating hamster exhibited higher than normal oxygen consumption rates after freezing. This result did not occur with slices from the warm-adapted... 

Frozen fixed tissue is sectioned in a cryostat also known as a microtome in a freezer. The tissue is fixed in 4% paraformaldehyde, rinsed, and then cryoprotected by infiltration in 20% sucrose in buffer with agitation overnight at 4°C (cold room). After 24 h the tissue blocks will sink in the solution, indicating that they are infiltrated. This infiltration step is critical. If skipped, the tissue will freeze with damaged cells and holes from ice crystals, and the resulting tissue sections on microscope slides will be brittle and might crack. The tissue must be fixed first to hold the cells in place, as cryoprotection and freezing without fixation will destroy the tissue. 

Infiltration - processing of tissue blocks with by in 20% sucrose in buffer with agitation overnight at 4°C (cold room) to cryoprotected the tissue for freezing. 

Place the vial containing acetone and tissue in the refrigerator to raise the temperature. The tissue can now be directly stored (see Note 22), infiltrated for resin embedding, or rehydrated. After rehydration, it can be processed for immunohis-tochemistry as a wholemount, or cryoprotected and sectioned in a cryostat, prior to ICC (see Sections 3.3. and 3.4.)... 

Cryoprotect the fixed tissues by immersing them in sucrose solution at 4 C overnight, or until the tissues sink to the bottom of the sucrose solution (see Note 22). 

To minimize cellular destruction by ice crystals, the tissues must be cryoprotected prior to sectioning. The presence of high concentrations of sucrose results in the slow formation of small ice crystals, rather than the rapid formation of large ice crystals. 

Cryoprotect the postfixed tissue (Section 3 6.1., step 5) by overnight immersion m ice-cold cryoprotection solution. 

Steinbrecht, R. A. (1980) Cryofixation without cryoprotectants. Freeze substitution and freeze etching of an insect olfactory receptor. Tissue and Cell, 12, 73-100. 

Cryoprotectant Substance that protects biological tissue from freezing damage. 

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