DAB

In most chromogenic IHCLoading..., the detection system carries horseradish peroxidase (HRP) to the bound antibody. Given hydrogen peroxide, HRP oxidizes 3,3′-diaminobenzidine into a brown polymer that precipitates at the site. The precipitate is insoluble in water, alcohol and xylene, so it survives dehydration, clearing and permanent mounting, and it does not fade.

Its color is a broad, featureless brown that absorbs across the visible range, most strongly in the blue. Adding metal salts such as nickel or cobalt during development turns it gray to black, which is used for a second marker or for contrast. That changes its color, so the stain vector used for color deconvolutionLoading... must change with it.

It is tempting to read DAB optical densityLoading... as a measure of how much antigen is present. Three things break the link. The signal is enzymatic: each HRP molecule deposits a great deal of product, the reaction depends on time and temperature, and it saturates at high antigen. The precipitate scatters light as well as absorbing it, which violates the Beer–Lambert assumption that optical density is proportional to amount. And the brown has no absorption peak, so it is harder to separate cleanly from hematoxylinLoading....

DAB intensity is therefore meaningful within one assay run, on one instrument, against controls, and is best used for positive area, cell counts or scored categories rather than absolute concentration.

Because DAB absorbs most in the blue, strongly stained areas are darkest in the blue channel, so that is the channel carrying the most information and the least light. Anything that adds to or subtracts from blue shifts the DAB estimate directly. A yellow color castLoading... from a stale white balanceLoading... reads as extra DAB everywhere; vignettingLoading... makes corner cells look more stained; and clippingLoading... or an unsubtracted camera offset distorts the darkest, most strongly stained pixels.

A practical rule: balance on blank glass, correct with a flat fieldLoading... and a dark frameLoading..., and include a positive and a negative control slide in every imaging session.