I’m trying to detect KI67 in rat bone. Have someone have working protocol for doing this?
Thanks in advance
to detect DAB from a file you can use different method to split your color like RGB or use the pluggin color deconvolution.
Note: A very frequently asked question relates to quantification of immunostain intensity (for example DAB intensity) to evaluate antigen expression. However, one needs to consider some important issues that prevent doing this in a quantitative manner:
- 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. 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).
- 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.
- The chromogen DAB does not follow Beer-Lambert law either. See, e.g., the paper by CM van der Loos:
“The brown DAB reaction product is not a true absorber of light, but a scatterer of light, and has a very broad, featureless spectrum. 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. As a consequence, darkly stained DAB has a different spectral shape than lightly stained DAB.”