Titlebook: Confocal Microscopy; Methods and Protocol Stephen W. Paddock Book 19991st edition Humana Press 1999

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by imaging only one plane within the sample at a time so that variations in depth can be quantified (.). This has both positive and negative aspects. The advantage is that a series of such slices can be reconstructed to give 3D views and enable volume analysis of the sample, and that any one slice

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detection methods (.). However, this method of detection until recently has been limited by the required use of alkaline phosphatase conjugated secondary antibodies and chromogenic substrates. The use of alkaline phosphatase substrates and their diffusible products limits the resolution of staining

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st cells produced by animals, and sperm are frequently the smallest. Observation of the initial steps of fertilization, including sperm-egg binding and fusion, can be challenging owing to the size discrepancy between these two cell types and, in many species, the rapid time course over which they oc

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zation in developing embryos. For . researchers, the ease of generating such antisera (.) and the number and widespread availability of existing antibodies make immunofluroescence of embryos an indispensable technique. The use of fluorochrome-conjugated secondary and/or tertiary antibodies on . embr

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which prevents frozen sectioning because the yolk crystallizes and tears the sections. In addition, the yolk autofluoresces, making whole-mount immunocytochemistry possible, but difficult due to background from out-of-focus fluoresecence. All of these problems can be solved through confocal microsc