Frozen specimens

HPLC The compound F obtained is further purified on a column of TSK DEAE-5PW, using 40% aqueous acetonitrile containing 85 mM NaCl and 3 mM NaHCC>3. The final yield of F is about 2 mg (purity 80-90%) from 500 g of frozen specimens. A small amount of 2-mercaptoethanol added at the last desalting step may significantly improve the purity of the final product. 

Purification of Latia luciferase and the purple protein. According to Shimomura and Johnson (1968c), frozen specimens of Latia (10 g) are vigorously shaken in 200 ml of cold 5mM sodium phosphate buffer (pH 6.8) for 15 minutes. Latia luciferase is extracted into the buffer and the solution becomes turbid. Four batches of such turbid solutions are combined and centrifuged, and the clear supernatant is... 

Frozen specimens of O. enopla (lOOg) were treated with one liter of boiling ethanol for 1 minute to destroy enzymes. The mixture was... 

Another microscopic technique is to freeze the specimen and then fracture it with a knife. A knife cutting through the frozen specimen splits the membrane down the middle, exposing the inside of the bilayer (fig. 17.13a). If the Davson-Danielli model for membrane structure were correct, the two exposed surfaces would be featureless. However, electron micrographs of metallic casts of such samples reveal surfaces studded with particles of various sizes (fig. 17.13(f)- Additional studies indicate that these particles are proteins that are deeply embedded in the membrane. The particles seen on the inner and outer leaflets of the bilayer usually differ in size and distribution because of an asymmetrical disposition of the proteins across the bilayer. 

A tissue section cut from a frozen specimen in this situation, ice is the supporting matrix. See Yamada, E. and Watanabe, H., High voltage electron microscopy of critical-point dried cryosection, J. Electron Microsc. 26 (SuppL), 339-342, 1977 Maddox, P.H., Tay, S.K., and Jenkins, D., A new fixed cryosection technique for the simultaneous immuuocytochem-ical demoustratiou of T6 and SlOO antigens, Histochem. J. 19, 35-38,... 

Indeed, these situations can be observed in living cell structures. During electron microscopic observation of freeze-thawed cell nuclei, rapidly frozen specimens had the original tissue structure. When these specimens were thawed rapidly, no difference could be seen in comparison with the control. Only a minor increase in the affinity to dyes and a slight condensation of chromosome have been observed. On the contrary, when specimens are thawed slowly many large cavities are observed, which indicates that the cellular materials are forced out by ice crystals. In these specimens, a very serious rupture of nuclear membrane was also observed. Similar results have been obtained with freeze-fractured electron microscopic observation of rapidly frozen red blood cells. In these experiments, membrane structure had been damaged in the regions where ice crystals and red blood cell membranes were in close contact. 

Serum or heparinized plasma, free of hemolysis, should be used. Complexing anticoagulants — such as citrate, oxalate, and EDTA— must be avoided, because they bind cations, such as Mg and Zn % which are necessary cofactors for ALP activity measurement. Blood transfusion (containing citrate) causes a transient decrease in serum ALP through a similar mechanism. Freshly collected serum samples should be kept at room temperature and assayed as soon as possible but preferably within 4 hours after collection. In sera stored at a refrigerated temperature, ALP activity increases slowly (2%/d). Frozen specimens should be thawed and kept at room temperature for 18 to 24 hours before measurement to achieve full enzyme reactivation. 

Twenty-four-hour collections offer little advantage, delay diagnosis, and increase the risk of losses during the collection period. PBG and porphyrins are stable in urine in the dark at 4 °C for up to 48 hours and for at least a month at -20 Specimens for ALA estimation should be promptly refrigerated. Urine specimens can be stored at 4 C in the dark for at least 2 weeks without significant loss of ALA, and frozen specimens are stable for weeks. Whereas PBG is more stable around pH 8 to 9, ALA is more stable around pH 3 to 4, although more acidic environments notably reduce ALA stability. 

Thaw frozen specimens in front of a fan for a few minutes until they are at room temperature. Do not thaw frozen specimens in a refrigerator. 

In freeze-fracture preparations made without consideration of the residual gas composition in relation to specimen temperature, water vapor can condense on the frozen specimen. This can occur nonrandomly on the fracture surface, giving the impression of membrane particles that aren t there or masking those that are. 

For urine analysis sample aliquots are diluted 1 1 with distilled water before application to the AAS stabilized temperature platform (Leung and Henderson, 1982). Fecal analysis require considerably more complicated preparation steps than serum or urine. The procedure developed in the author s laboratory (Brown et al., manuscript in preparation) is summarized as follows Frozen specimens are thawed and distilled water is added (1 mL per 2 g feces) and the sample is homogenized in a sealed container on a paint shaker. A 10 mL aliquot is ashed at 550°C in a muffle furnace, dissolved in dilute HNO3 snd analyzed by GF-AAS. 

Bone samples for aluminium analysis have been taken from the iliac crest at the time of biopsy or at autopsy (Alfrey et al.. 1976 Maloney et al., 1982) and the specimen placed in an Al-free plastic container. Bone for histological staining is fixed in 10% buffered formalin (Maloney et al., 1982). Crapper et al. (1976) analyzed brain samples from specific areas of the cerebral cortex and from subcortical area. Alfrey et al. (1976) analyzed brain samples from frontal cortex. Whole brain as well as white and grey matter were analyzed. A description of how the specimen was handled before analysis was not provided. Crapper et al. (1976) transported and stored brain samples frozen in Al-free plastic containers and performed dissection from the frozen specimen in a dust-controlled room. All instruments and gloves were rinsed in aluminium-free water. At frequent intervals, this entire procedure was performed on standard homogenized freeze-dried brain powder to ensure little or negligible aluminium contamination. 

Frozen specimens should be maintained in a frozen state as long as possible before arraying, and when defrosted, only warmed sufficient to produce a liquid state for mixing. If LCM-derived specimens are used, they should be maintained either at 4°C or frozen till ready for arraying. Even lysates derived from formalin-fixed paraffin embedded tissue should not be left at room temperature after extraction. 

Figure 9.13 Cold-stage system for fracturing frozen specimens, etching the fracture surfaces by sublimation, and coating them with a few nanometers of conductive metal...

Approximately 20-60 mg surgically resected glioma tissues were frozen in liquid nitrogen at — 170°C. Prior to HRMAS H-MRS, the frozen specimens were washed in 0.9% physiological saline containing heavy water (D20). HRMAS H-MAS was performed on a 600-MHz spectrometer with a microprobe at room temperature, and the spectrum analysis was performed with Xwin-NMR software. 

Figure 5 (a) Frozen specimen preparation machine, (b) inside... 

Frozen specimen preparation machine (for example, JEOL JFD-9010)... 

The frozen specimen can be kept in liquid nitrogen up to one year. 

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