Snap-freezing

Compound (Miles Laboratories, Elkhart, IN), snap-frozen, and cut into sections for comparison with paraffin-embedded cell sections (3) FFPE Cell Blocks Six cell pellets were fixed in 10% neutral buffered formalin immediately after harvest, at room temperature for 6,12,24h, 3,7, and 30 days, respectively. For further comparison with the cell model system, recently collected sample of human breast cancer tissues were processed by OCT-embedding and snap-freezing the corresponding routine FFPE block that was obtained from the Norris Cancer Hospital and Research Institute at the University of Southern California Keck School of Medicine (USC). This tissue block was processed routinely (formalin-fixed 24h and processed by automatic equipment). 

Air drying, usually with subsequent chemical post-fixation, is applicable for cell smears, cytospins and cryosections. Snap freezing (usually in liquid nitrogen) is routinely employed for tissue probes for subsequent cryosectioning. Chemical fixation is commonly carried out in aldehydes (e.g. formaldehyde) for tissue probes and cultured cell monolayers, and in acetone or alcohols (methanol or ethanol) for cryosections and cell preparations. 

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). 

For tissue collection, take samples according to the method of analysis snap-freeze portions of kidney in liquid nitrogen for RNA extraction, roll tissue in Tissue Tek OCT (Cryoform, IEC, Needham, MA) and snap freeze in isopentane cooled over dry ice fix tissue in 4% paraformaldehyde for in situ hybridization or in 10% buffered formalin for routine histology. 

With the exception of vacuum freezing (or snap -freezing), the initial cooling is always done at atmospheric pressure, sometimes in a separate cabinet. 

Remove supernatant and snap-freeze the pellet by placing in liquid nitrogen or dry-ice store at -70°C until required. (The enzyme should be stable for up to 3 mo.)... 

Tissue snap-freeze (dry-ice or liquid nitrogen) and store at -20°C or -70°C. Lymphocytes collect whole blood into universal containing EDTA ( final concentration 25 mM). Isolate lymphocytes by density centrifugation (12), wash with PBS and store cell pellet at -20°C. 

Once a distinct color change has been observed, transfer the entire contents of the broths into 1 mL cryotubes, assign a batch number, and snap-freeze in the vapor phase of liquid nitrogen. 

The lipid was hydrated to a concentration of 20-mg/ml. The formulation was to be lyophilized (freeze-dried) by snap freezing in liquid nitrogen and sublimation of the water, resulting in dry and stable lipid vesicles. 

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. 

Keightley, D. D., Tilley, W. D., and McK. Cant, E. L., Effect of snap freezing, dithiothrestol, and storage on estimations of estrogen receptor sites. Clin. Chim. Acta 88, 337-343 (1978). Kennedy, B. J., Fluoxymesterone therapy in advanced breast cancer. N. Engl. J. Med. 259, 673-675 (1958). 

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. 

Transfer chromatin from dialysis membrane into polypropylene round-bottomed tube. Spin at 12,000g for 10 min in SS-34 rotor. Transfer chromatin into a new tube and divide into 300-pL aliquots (each of which should contain 300-500 pg) in Eppendorf tubes, snap freeze in liquid nitrogen and store at -80°C or use immediately for immunoprecipitation. 

At the end of a topical treatment, samples in addition to perfusate may be collected to determine the amount and distribution of penetrated compound within tissues under the topical dosing site. The most precise technique available for this purpose is to take a core biopsy through the dosing site, snap freeze it in liquid nitrogen, and then cut serial sections to precisely localize chemical distribution within the skin as a function of penetration depth. In these smdies, which are fully described in the literamre (Riviere, Monteiro-Riviere, et al., 1992 Monteiro-Riviere, Inman, et al., 1993), the surface of the appUcation site is first gently washed with a mild soap solution and then dried with gauze. Cellophane tape is then applied to... 

Collect the clear extract by side puncture of the centrifuge tube, snap freeze in aliquots in liquid nitrogen, and store at -80°C. 

Centrifuge the cells for 20 min at 4000 rpm and 4 °C. Decant medium. Resuspend each pellet in 5 mL 2YT medium. Pool the supernatants containing the bacteria and add 0.5 volume of 50% glycerol in H2O (v/v, sterile filtered on a 0.22 pm membrane) to obtain a final concentration of 16.5% glycerol in 2YT medium. Measure the OD oo of the final solution. Prepare 750 pL aHquots and snap freeze them on dry ice prior to store them at — 80 °C. 

Hold the cork disk with forceps and lower it quickly in the melting Arcton (isopentane) to snap-freeze the tissue. 

Note It is recommended to continue with the trypsin digestion step immediately to minimize protein degradation. If lysates need to be stored at this point, then snap-freeze them using a dry ice/ ethanol mixture and store them at -80°C. 

Snap-freeze the sample in liquid nitrogen, and store at — 70°C. 

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