Permanent preparations

Other authors have proposed that masturbation occurs because sperm have a limited shelf life (Levin 1975). Here, however, the hypothesis is that masturbation produces a more competitive EPC ejaculate. If so, it could imply that, because of their high ejaculation rate, bisexuals are permanently prepared for EPCs whereas heterosexuals anticipate each event opportunistically. In which case it remains possible that, as hypothesised, bisexuals are a morph adapted to an above average involvement in sperm competition (section 5.2). 

There are a few common enzymes that have been employed in these types of assay systems over the years, the chief among them being the peroxidase enzyme (3). Peroxidase has an oxidative function when in conjunction with a source of oxygen, transferring electrons to a molecule, which becomes oxidized. The peroxidase enzyme found in the horseradish plant has been used for its ability to carry out this function, for the fact that it is easily obtained, and for the antigenic differences from most mammalian forms of the enzyme. The oxidative function of this enzyme allows for the use of chromogens, which when oxidized, not only change color, but precipitate in such a manner as to render a permanent preparation. 

Rapid. Permanent preparations. Simple nuclear counterstains may be used for studying tissue architecture in relation to immunolabeling. 

Relatively rapid. Sharp, black, high-resolution reaction product. Sensitive. Permanent preparations. Nontoxic reagents. Can be studied using various forms of microscopy including dark ground, epipolarization, confocal laser scanning and electron microscopy. 

Direct immunoenzyme. Rapid. Permanent preparations. Simple nuclear counterstains may be used for studying tissue architecture in relation to immunolabeling. Use of toxic chromogens is involved. Need to block endogenous enzyme activity. Extravagant when more than just a few target antigens are studied. 

Produce permanent preparations by mounting in an aqueous-based medium, such as Immu-mount, or in a xylene-based mounting medium, such as Fluoromount, following dehydration through an ethanol senes (i.e., 5 min each in 10, 20, 30, 40, 50, 60, 70, 80, and 90% ethanol in distilled water followed by three 5-min steps in absolute ethanol see Note 4). 

In preliminary experiments it may often be more important that a rough estimation of the effectiveness of the chemical can be quickly obtained than to have good permanent preparations for a detailed analysis. In such cases, the acetic-orcein method of Tjio and Levan (1950) is useful. 

HRP is not a fluorescent tracer (it generates a dense brown reaction product as outlined below), but has the advantage of producing permanent preparations and can be used in combination with whole-mount in situ hybridization techniques. 

Permanent preparations Water-soluble permanent mountants (i.e., which set) usually suffer from shrinkage, which can ruin a specimen. The more viscous they are, the less of a problan this is likely to be. However, viscosity makes the mountant hard to handle. Probably the best aqueous, setting mountant is Gelvatol (Mon-santo)/Mowiol 4-88 (Hoechst), which hardens overnight and can be stored as frozen aliquots (see section 2.4). 

Oldest of all the histochemical tests is the iodine reaction 60) with starch, applied to plant histology in the early nineteenth century by Caventon and Raspail. For almost all purposes, it has been replaced by methods that give better color and contrast, and more permanent preparations. Still, the iodine reaction is used in a modem histochemical method for phos-phorylase (54)-... 

The preparations can be stored or stained immediately with lactic-acetic-orcein, toluidine, or Unna blue. Toluidine blue is generally preferred for immediate examination of nonpermanent preparations. Lactic-acetic-orcein and Unna blue are used for finer examination and for permanent preparations. 

Glass coverslips (22 X 22 mm also called cover glasses) siliconized if the coversKp is to be removed for a permanent preparation or if the chromosomes are to be used for another procedure, such as in situ hybridization (for preparation of siliconized coverslips, see below). 

Secondary antibody. We prefer to use a directly conjugated secondary antibody for detection, whether the detection is by diaminobenzidine (DAB) or by confocal fluorescence microscopy. To obtain a permanent preparation, we use HRP-conjugated goat anti-mouse or anti-rabbit antibody (at a dilution of 1 200 Bio-Rad). For visu-... 

The subsequent introduction of immunoenzyme techniques [6, 7], primarily the horseradish peroxidase (HRP) method developed in the 1960s by Nakane [8,9], represented a significant boost to the use of ICC, as it became possible to obtain permanent preparations that could be observed with a conventional light microscope. Therefore, ICC not only started diffusing among basic scientists but also began to be employed for diagnostic... 

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