In situ macromolecular analyses typically have five steps. The tissues must be fixed, permeabilized, and probed for the molecule of interest. RNA species can be detected using tagged DNA or RNA probes. Protein species, in contrast, are typically detected using a tagged antibody. The samples are then mounted onto slides and analyzed microscopically. In situ methods have advanced through improvements in microscopy technologies, such as confocal and deconvolution microscopy. Traditionally, microscopic analysis of thick tissues could only be accomplished using sectioning of paraffin-embedded samples. However, confocal and deconvolution techniques allow the user to make optical sections through a thick sample, improving the resolution and removing fluorescent background from sections above and below the focal plane without introducing the artifacts that can occur with manual tissue sectioning. These techniques also allow the user to reconstruct the original three-dimensional image, so that staining can be viewed in the context of the entire ...
A Look at Drosophila Pattern Formation
Researchers interested in gene expression studies adopt one of two approaches. They can either examine the expression of a given gene in a population of cells in aggregate, or they can study the gene on a cell-by-cell basis in situ. The advantage of the former approach is its simplicity: It is generally easy to prepare RNA or protein from a given tissue sample and to probe it for the gene or protein of interest. But there are several disadvantages associated with the population approach. First o
Written byJeffrey Perkel
| 9 min read
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