Tissue freezing medium

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). 

Mount the tissue block on the cryostat stub with embedding medium such as Tissue-Tek O. C. T. Compound (SakuraFinetekU.S.A. Inc., Torrance, CA)orTBS Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC). 

Two samples (indented and spherical mannitol particles) were suspended in tissue freezing medium on a sample holder (—25 °C) and sectioned (5 pm thickness) using a cryomicrotome (CryoStar NX 70, Thermo Scientific, Waltham, MA, USA). Process temperature was set to —24 C for the cutting process. The cross sections were then placed on an SEM sample holder and prepared for SEM analysis as described in Sect. 2.2.7. 

To freeze uninfected and infected HL60 cells, transfer media and cells from tissue-culture flask into 50 mL conical tubes. Centrifuge at 450 x g for 5 min to pellet cells. Decant or aspirate supernatant and discard. Resuspend 1 mL of cells at a final concentration of 5 x 10 cells/mL in freezing medium. Transfer contents ( 0.5 mL per tube) into NUNC cryovials. Freeze at -80 °C overnight, then transfer vials to Uquid nitrogen the next day. Freeze infected HL60 cells at 90% infection level. 

There has been an increase in the use of cadaveric heart valves for patients with valvular defects. The valves are best stored by freezing but some success has been achieved by simple cold storage in an antibiotic medium made up of ingredients common to most tissue culture solutions. At a storage temperature of 4 °C there is a continual loss of viability of fibroblasts so that by three weeks there are practically no viable cells and the valves cannot be used. 

For cryosectioning, tissue samples are quickly frozen with or without freezeembedding medium (e.g., Tissue Tek Miles Laboratories) and stored at 80°C until analysed. Optionally, aldehyde prefixation can also be used for tissue and organ probes before snap-freezing. Cutting of frozen tissue blocks is performed with a cryostat (a microtome mounted in a freezing cabinet). 

Small pieces of tissue (approx. 5 mm) are quickly frozen with or without freeze-embedding medium (e.g.,Tissue Tek Miles Laboratories) and stored at 80°C until analysed. 

Immerse the tissue blocks in an embedding medium, and freeze quickly with crushed dry ice. The frozen tissue blocks can now be stored at —80°C. 

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. 

Kaeser, W., Freeze-substitution of plant tissues with a new medium containing dimethoxypropane, J. Microscopy, 154, 273-278, 1988. 

Small pieces of tissues are fixed in PLP for 4 h at 4°C on a rotatory shaker, washed overnight at 4°C in 100 mM phosphate buffer, pH 7.4, containing 10% sucrose, followed by several washings 15% sucrose in buffer (overnight at 4°C), 20% sucrose in buffer (4 h at 4°C), 20% sucrose plus 20% glycerol (1 h at 4°C). The pieces are embedded in OCT medium (Miles), in a small cup of heavy aluminum foil, by snap-freezing in ethanol-dry ice bath while avoiding contact between ethanol and OCT. Frozen sections are cut, 6-12 pm thick, mounted on albuminized slides and washed 3 times 10 min with PBS-10% sucrose. 

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