Good counterstain for DAB based IHC in brain tissues

The fading of NiDAB is due to one of two features. 1) if you keep ANY bleach in the room, the slightest exposure of a slide or glassware or air with some bleach will cause NiDAB to fade (we do not allow ANY bleach in our laboratory but let others nearby keep it. 2) NiDAB will fade if beakers, slides, or any other glassware used in the staining came into contact with Alconox washing solution. NEVER NEVER use the powdered glasswashing material if you plan on using NiDAB. Instead, the liquid diswasher material cleans just as well but keeps the NiDAB from fading. A third consideration is seen only when NiDAB is used in concert with immunofluorescence. While we have conducted many studies with nuclear signals stained with NiDAB (including BRdU) we found that water soluble media cannot be used but instead, the NiDAB and fluorescence are maintained for YEARS when slides are dehydrated and coverslipped with xylene based mounting media (low fluorescent Permount for example, or Krystalon). Then you get the best of both worlds: maintenance of the NiDAB signal and long term storage of the fluorescence. With BRdU this approach in neurons enabled good neuron nuclear labeling for cells not containing the BRdU, and for beautiful BRdU. I do not like cresyl violet but would recommend another stain, Neutral Red which gives much better contrast to the NiDAB product.

One more feature: when using DAB products (brown or Black) one needs to select the color of the counterstain that is on the opposite side of the spectrum: DAB alone - methyl green is optimal (the hematoxylin or other blue stains can be slightly purple making the distinction difficult; for NiDAB as I mentioned, the RED counterstains are optimal and work wonderfully - giving as clear cellular detail as hematoxylin. It is not acidity but "reducing agents" that cause NiDAB to fade.

Souce: NovoPro    2018-05-08