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 on the spot. Water, alcohol and xylene do not dissolve the precipitate, so it survives dehydration, clearing and permanent mounting, and it fades less quickly than the products of other precipitating peroxidase substrates.

The brown is broad and featureless, absorbing across the visible range and most strongly in the blue. Nickel or cobalt salts added during development turn it gray to black, for a second marker or for contrast. The color has changed, so the stain vector used for color deconvolutionLoading... must change with it.

DAB optical densityLoading... is often read as a measure of how much antigen is present. Three things break that link. The signal is enzymatic, and its product clogs at high staining intensities, so the darkest areas stop keeping pace with the 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....

So DAB intensity is meaningful within one assay run, on one instrument, against controls. Use it for positive area, cell counts or scored categories, not absolute concentration.

DAB absorbs most in the blue, so strongly stained areas are darkest in the blue channel. That channel carries the most information. With a halogen lamp it also gets the least light, and anything that adds or removes blue shifts the DAB estimate directly.

A yellow color castLoading... from a stale white balanceLoading... reads as extra DAB everywhere. With vignettingLoading..., corner cells look more stained. An unsubtracted camera offset or clippingLoading... distorts the darkest, most strongly stained pixels.

The 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.