Dehydrated specimens

Dehydrate specimens with 100% ethanol, four changes for 2 min each. 

Completely water-swollen collagen differed markedly from the relatively dry [equilibrated at 50% relative humdity (RH)] protein (8) in its wettability by water-immiscible liquids. In the water-swollen case, the apparent critical surface tension diminished to ca. 30 dynes/cm, indicating that a protein interface in nature is not correctly modeled by dehydrated specimens. 

The critical point refers to the certain combination of temperature and pressure at which the liquid density is equal to the vapor density. At its critical point, liquid will become vapor and is easily removed. We cannot directly remove water using its critical point because the temperature and pressure of the critical point (374°C and 22 MPa) are too high and may damage the specimen. Alternatively, we can replace water with a transitional fluid that has a critical point with lower temperature and pressure. Liquid CO2 or Freon is often used as the transitional fluid. The critical point for liquid CO2 is 31.1 °C and 7.4 MPa. The common procedure is described as follows. First, water content in a specimen is removed by dehydration with an ethanol series (30, 50, 75 and 100%). Then, the dehydrated specimen is transferred into an ethanol-filled and cooled chamber in the critical-point drying apparatus. The transitional fluid is introduced until it completely displaces ethanol in the chamber. The chamber is gradually heated and pressurized to reach the critical point of the transitional fluid. After reaching the critical point, the transitional fluid vaporizes, and this vapor is slowly released from the chamber until atmospheric pressure is reached. Then, we can retrieve the intact, dry specimen from the chamber. 

Dehydrated Specimens. Ideally, the water molecules in a zeolite exist as discrete molecules and can be removed easily by heating. In... 

Wash the slides three times in PBS and treat the slides with 0.2 M HCl for 10 mm. Continue to the ISH protocol (see Subheading 3.3.1., step 4) or dehydrate the slides through increasing concentrations of ethanol (50-100%) and store the dehydrated specimens in a light-proof box at room temperature before treatment with proteinase K (see Subheading 3.3.1., step 4)... 

To dehydrate specimens, replace the 70% ethanol with absolute ethanol. Leave for 10 minutes, then remove the ethanol, and add fresh absolute ethanol. 

It is relatively simple to analyze a specimen for a host of components, as compared to the obtaining of the specimen in a dehydrated infant, particularly one who is in circulatory collapse. 

Fixation tissue samples for immuno histochemistry were fixed in 2% paraformaldehyde, 0.25% glutaraldehyde and 3% sucrose buffered with 0.05M phosphate buffer pH 7. After incubation for 2 hours at 25 °C and 63 hours at 5°C the specimens were washed 3 x 20 min. in phosphate buffer pH 7. Dehydration was carried out using series of ethanol washings 50, 70, 80, 96% followed by 3 x in 99% (V2 hr in each). After additional treatment with 2x2 hrs in petroleum ether (shellsol D70k, Q7712) and 2 x 2 hrs in paraffin with 7% beeswax, the samples were embedded in paraffin. Cross sections of 12.5 /im were made on a Supercut 2050 Reichart Jung pyramitome. 

III. Transmission electron microscopy of radish seeds Transmission electron microscopy (TEM) of radish seeds was done as listed below For TEM preparations, the specimens after fixation and dehydration, were embedded in Epon 812 resin (Luft, 1961). Thick sections (ca. 1mm each) were stained with 0.1% toluidine blue and observed with a Zeiss light photomicroscope. Thin sections, obtained with a diamond knife on a Supernova microtome, were sequentially stained at room temperature with 2% uranyle acetate (aqueous) for 5 min and by lead citrate for 10 min (Reynolds, 1963). Ultrastructural studies were made using a Philips CM12 transmission electrone microscope (TEM) operated at 80 KV. 

Glauert, A. M. (1975). Fixation, Dehydration and Embedding of Biological Specimens. North-Holland, Amsterdam,the Netherlands. 

Finally, the localizations of low-molecular-weight compounds requires special specimen preparation techniques, as these compounds are often diffusible, water- or organic-solvent soluble, and solubilized by conventional fixation and dehydration procedures. The reader is referred to ref. (12) for the processing of cells and tissues for the cytochemical and histochemical localization of these compounds. 

Specimens were dehydrated by the standard ethanol series as described. 

Note We recommend that after performing step 1 above that the specimens be rinsed three times in a phosphate buffer (pH 7.2), which will remove any residual glutaraldehyde before beginning the dehydration series. Garduno etal. (13) also use a second technique in which they cut small pieces of medium containing diatoms and immediately fix the pieces in liquid nitrogen. The pieces are freeze-dried and then gold sputter coated. 

Each specimen was dehydrated, infiltrated and embedded in Technovit based methylmethacrylate. One section was cut and around in preparation for scanning electron microscopy (SEM). In each case, three overview photos were necessary and four high magnification fields (40X) were photographed and digitized. These fields were later analyzed for volume fraction of soft tissue, bone... 

In most cases, fixation may be carried out at room temperature. Duration of formalin fixation depends on the nature and the size of the specimen, and may vary from 15 min to 24 h. Longer fixation may be associated with a partial loss of the antigenicity of the component of interest. After formalin fixation, tissue samples are washed in three changes of the buffered saline (PBS) from 15 min to 2 h, but not longer than 24 hours on the whole, since the formaldehyde fixation is partially reversible. After washing in PBS, specimens may be either snap-frozen in liquid nitrogen for subsequent cryosectioning, or dehydrated and embedded in paraffin or synthetic resin. 

Tissue processing tissue specimen (0.5 1.0 mm3) are fixed in 4% buffered formalin for 30 60 min, post-fixed in 1% osmium tetroxide in cacodylate or phosphate buffer, pH 7.2 7.4, stained en bloc for 30 min with 2% aqueous uranyl acetate, then dehydrated in ethanol and embedded in epoxy or acrylic resin. 

Specimens were post-fixed in 1% OSO4 in sodium cacodylate buffer, dehydrated in ascending alcohol solutions, and embedded in Epon LX 112. Semi-thin sections (1.0 pm thickness) were cut with a diamond knife and stained with Richardson s dye (contains methylene blue) or PAS-reagent. Micrographs were made with an Olympus New Vanox microscope. 

Glauert, A. M. (1974) Fixation, dehydration and embedding of biological specimens, in Practical Methods in Electron Microscopy, vol. 3 (Glauert, A. M., ed.), North-Holland, Amsterdam, pp. 1-201. 

Continue with Subheading 3., step 18 in the method. The positive reaction product with this chromogen will be red, with the nuclei a light blue. Rather than dehydrating the specimens in ethanol and xylene, allow them to dry, add 1 drop of Crystal/Mount (Biomeda Corp, Foster City, CA) to the specimen and bake them in a 60°C oven for 30 min. This preparation is permanent and can be cover-slipped with Permount if needed. The Crystal/Mount will form a hard plastic coating on the slide, but it can be damaged by smudging. 

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