As a consequence, darkly stained DAB has a different spectral shape than lightly stained DAB. This means that DAB does not follow the Beer-Lambert law, which describes the linear relationship between the concentration of a compound and its absorbance, or optical density. A hemocytometer is most frequently used to perform this task, and cells need to be manually counted on eight 1 × 1-mm areas on the two panels of the hemocytometer. “The brown DAB reaction product is not a true absorber of light, but a scatterer of light, and has a very broad, featureless spectrum. Counting mammalian cells before transfer into plates or dishes is an important task in cell biology. See, e.g., the paper by CM van der Loos : The chromogen DAB does not follow Beer-Lambert law either.Immunohistochemistry uses a series of amplification steps to visualise the results making it difficult to control what the final intensity of the amplified signal actually represents in terms of amount of antigen.In fact most histological stains are non-stoichiometric (some exceptions are Feulgen stain which is commonly used for DNA cytometry and phalloidin for visualising actin). ![]() Antigen-antibody reactions are not stoichiometric, therefore “darkness of stain” does not equate to “amount of reaction products” or to “expression” of a given antigen.However, one needs to consider some important issues that prevent doing this in a quantitative manner: ![]() ![]() Note: A very frequently asked question relates to quantification of immunostain intensity (for example DAB intensity) to evaluate antigen expression. To detect DAB from a file you can use different method to split your color like RGB or use the plugin color deconvolution.
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