Preparation of the Slide

The following is an example of the steps required for measuring the in vitro motility of actin filaments over a surface of monomeric smooth muscle myosin. 

Myosin at concentrations between 30 and 200 p.g/ml in 0.5 M NaCl, 10 mM MOPS (pH 7.0), 0.1 mM EGTA, 1 mM DTT (buffer B) is introduced into the flow cell, which is inclined at an angle of about 30°. 

After about 60 sec, the flow cell is washed with 2 to 3 vol of buffer B containing 1 mg/ml bovine serum albumin to remove unbound myosin and to block the surface in order to prevent nonspecific binding of actin. 

Two vol of 20 nM rhodamine phalloidin labeled actin in buffer B is used to wash the flow cell. 

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In this chapter, we provide technical details regarding the construction of a robotic device capable of spotting DNA with the resolution required for microarray experiments, as well as protocols for preparation of the slides, spotting of the DNA, preparation of fluorescent probes, hybridization, and washing of slides. Available methods of scanning are also discussed. 

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. 

In the preparation of smears, it is necessary to get a uniform thin film to avoid cells bunching up in layers. Start with a smaller sample amount if this is occurring. The cells will spread out over the surface of the slide and form a film with a feathered edge, if done properly. 

Preparation of the Au-Coated Glass Slide with Reactive Succinimide Esters... 

STEP 2 PREPARATION OF THE substrate(s). While ahnost any substrate can be used, we will use glass microscope slides in this example this is a common substrate and makes it easy to see the CdS film. The microscope slide can be cut to whatever size and shape is convenient. The slide should be cleaned well, since films usually do not adhere well to dirty surfaces. Suitable cleaning agents are trichloroethylene or/and sulphochromic acid, and the slide should be well rinsed with pure water. If the slide is clean, water dropped onto it will form a film (hydrophilic surface), while on a dirty (hydrophobic) slide the water will form drops. Needless to say, the part of the slide where deposition is to occur should not be touched with the hands after this treatment. 

Fig. 4 Slide preparation and probe immobilization scheme. A clean slide is treated with mercaptosilane (MPTS), and an intermediate mercaptosilane layer is formed on the surface of the slide. 5 disulfide-modified probes are immobilized onto the slide through thiol/disulfide exchange reactions...

Fig. 9.4. Preparation of the egg for the CAM assay. On day 3 of incubation, 2-3 ml of albumen are aspirated at the acute pole of the egg (a) to detach the developing CAM from the shell the holes are closed with plaster. The upper surface of the egg is brushed on with paraffin (b) and cut with scissors kept parallel to the surface so as not to damage the embryo (c). The window is covered with a glass slide and sealed with paraffin (d).

ASA films, such as Kodak Tri-X Pan for black and white prints or Ektachrome for color slides, are suitable for many applications. These films can be exposed at 800 or 1600 ASA and push-processed in a high-contrast developer. A combination that has found favor in many laboratories is that of Kodak Tri-X with the Diafine two-bath developer (1), which provides an ASA of 1600. In general, black and white film has a finer grain and is suitable in most situations, the exceptions being doublelabeling procedures in which both fluorophores are to be photographed simultaneously or the preparation of color slides for presentations. 

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