DAB tetrahydrochloride dihydrate is widely employed as a staining agent in histochemical (HC) and immunohistochemical (IHC) applications, where it plays an important role in peroxisomal staining procedures and in the detection of mitochondrial cytochrome c oxidase activity.

DAB tetrahydrochloride dihydrate, as a dye, is converted into an insoluble polymer upon oxidation, forming a dark brown precipitate in the reaction area, thereby enabling the visualization of target molecules. In immunohistochemistry, DAB tetrahydrochloride dihydrate serves as a substrate for peroxidase conjugated to primary or secondary antibodies. In histochemical staining Methods, DAB tetrahydrochloride dihydrate can be used to stain the activity of peroxisomes and mitochondrial cytochrome c oxidase (COX) in organelles [1].
Cytochrome c oxidase (COX) histochemical staining protocol [1] (This protocol is for reference only and can be adjusted according to specific experimental requirements):
1. Incubate the sections with ~50 µL COX staining solution (0.1 M NaPO4, 4 mM DAB tetrahydrochloride dihydrate, 100 µM cytochrome c, 2 mg/mL catalase) at 37°C for 45 minutes.
2. Wash three times with PBS, 5 minutes each, followed by sequential treatment with 75% and 95% ethanol for 1 minute each, and finally dehydrate with 100% ethanol for 10 minutes.
3. After staining, the samples must be completely dried before sampling.