Permanent Slides

A defined number of seeds of each plant was germinated in Petri dishes kept in a Phytotron growth chamber at 21 1°C in the dark. Root tips ( 2 mm) were collected after 5 or 7 days of germination on dependence on the plant species, and subjected to Feulgen staining procedure before the preparation of permanent slides for the observation at an Olympus CX40 microscope. 

As in the preliminary experiment, root tips were collected after 5-7 days, subjected to the Feulgen staining procedure as described above, then prepared adequately as permanent slides, and finally subjected to microscope observation. For each treatment, fifteen root tips (5x3 replicates) were prepared and 30,000 cells (2,000 cells per root tip) were examined in the case of MH, and 150 metaphases (5x3 replicates) were examined in the case of COL. 

A representative section (one fourth wedge) of the Millipore filter, with dust deposition side up, was placed on each of the pads for two minutes. Between each treatment the bottom side of the filter was blotted on a paper towel to remove access solution. After the final treatment the filter was dried for one hour at roan temperature. The dry, stained filter was then placed on a clear microscope slide and made translucent with immersion oil. Permanent slides were made by sealing the coverslips to the slides with clear fingernail polish. 

Permanent slides may also be produced by using the alternative combinations of Canada balsam or polystyrene in xylol, dammar in turpentine, gum arabic in glycerin, styrex in xylene, rubber in xylene and gelatin in water [33]. With a 1% solution this may be formed by dropping... 

For most purposes, a simple fixative such as acetic alcohol gives quite satisfactory results. The fixative consists of three parts 100% ethyl alcohol and one part glacial acetic acid. The best results are obtained when the fixative is freshly prepared and cool (4-10°C). The fixation is completed in 15 min, but we have found that the preparation of permanent slides can be facilitated by leaving the roots in the fixative for 2-24 hr at refrigerator temperature. If kept in a freezer, roots can be left in the fixative for a longer time than 24 hr. 

Staining procedure When dry, invert into a drop of 1.0% acetic orcein (0.5 g orcein in 45 ml glacial acetic acid reflux % hr, add 55 ml distilled water while warm, reflux % hr, let stand 24 hr, filter, and filter fresh before using) on a clean slide, suck out excess out excess stain with filter paper, and seal with Kroenig s cement. If permanent slides are desired treat dry coverslip preparation in following sequence 1.0% acetic orcein (30 min), 45% acetic acid (dip until free of excess stain), tertiary butyl alcohol equal equal xylene (1 min), xylene (1 min), xylene (1 min), and invert wet into Permount (thinned with xylene) on clean slide. 

For SEM investigations, specimens were stained with 2% osmium tetroxide, dehydrated in ethanol and acetone, critical-point dried and sputter-coated with gold, and studied at 20 kV with a Hitachi S-4000 scanning electron microscope. The liquid-fixed material and the permanent slides of serial microtome sections are deposited at the Institute of Systematic Botany of the University of Zurich (Z). 

To prepare fine museum-quality, permanent slides, it is best to mount specimens in Canada Balsam. Canada Balsam is usually supplied as a solution in xylene, although it is sometimes supplied solid and must be dissolved in xylene ( 60-65% [w/v]) before use. With use, the concentration of xylene in a stock of Canada Balsam decreases, and it is necessary to add more xylene. It is difficult to give precise recipes for Canada Balsam, because every user seems to prefer a slightly different viscosity. We tend to use a rather dilute solution of Canada Balsam so that it spreads easily and does not dry too rapidly while mounting specimens. The disadvantage is that there is actually less Balsam in a drop of the solution, and when dried, it may contract from the sides of the coverslip, sometimes even disturbing the specimen. Unfortunately, there is no substitute for experience when using Canada Balsam. 

Polymers will be elastic at temperatures that are above the glass-transition temperature and below the liquiflcation temperature. Elasticity is generally improved by the light cross linking of chains. This increases the liquiflcation temperature. It also keeps the material from being permanently deformed when stretched, which is due to chains sliding past one another. Computational techniques can be used to predict the glass-transition and liquiflcation temperatures as described below. 

To prevent this type of error, the balancer operators and those who do final assembly should follow the following procedure. The balancer operator should permanently mark the location of the contact point between the bore and the shaft during balancing. When the equipment is reassembled in the plant or the shop, the assembler should also use this mark. For end-clamped rotors, the assembler should slide the bore on the horizontal shaft, rotating both until the mark is at the 12 o clock position and then clamp it in place. 

Permanently stained slides may be mounted with a cover slip or may be air dried and examined after oil is added. Slides should be examined at a magnification of x400 to X500 or greater after they are scanned under lower power to find optimal areas. A x50 oil immersion objective is particularly helpful, as it allows the easy use... 

Permanently stained slides should be kept for 2 years. 

Permanence of Adsorption of BSA. By determining the total amount of protein adsorbed on the four sections (cf Fig. 1) of the glass slide (Table 1), it can be seen that within experimental error, that all the initially adsorbed BSA was accounted for... 

Walker PJ, Watts JMA. Permanent fluorescent test slides. J Microsc 1970 92 63-65. 

Alternatively, DNA on the slide can be hybridized with probe, followed by staining to effect chromosome banding. Fluorescence of the hybridized region can be photographed and mounted to serve as a permanent record. 

These slides are permanent and should not fade with time. If the presence of endogenons pigment is a problem with a particular specimen, or a color other than brown is desired as an indicator, different chromogenic compounds can be... 

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. 

Make slides permanent and suitable for viewing with an oil immersion objective by adding Permount and a cover slip. 

The question is similar to asking how many times one can strip and reuse a microarray before performance deteriorates. An alternative approach is provided by Hessner et al. (2003a) in which the cDNA probes are permanently labeled using fluorescein-labeled primers to the clone s vector insert region. Fluorescein is excited at 488 nm and emits at 508 nm, while Cy3 may be excited at 543 nm to reduce any spectral overlap with fluorescein. Thus, fluorescein-labeled cDNA probes may be printed down and the slide scanned for QC/QA purposes prior to hybridization. Since the same region is primer-labeled in each cDNA, a direct comparison between the relahve fluorescence units (RFUs) and the amount of cDNA probe can be defermined. 

Coverslip the slides with a permanent mounting solution, such as Histomount. 

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