Question.

I am doing free-floating immunohistochemistry with brain slices. The issue is that I always prepare my DAB (Hydrochloride sigma powder) aliquot it and store it in -20°C. But recently, once I thaw it the DAB quality decreases a lot, to almost not have any signal (I am used to almost black neurons with NiSO4). But all the protocols that I have found say that is okay to make aliquots and store them like that. Is there a stabilizer or something that I could use to make the stock solution last? or my only option is to prepare it always immediately before use?

Answer 1.

I do manual IHC-DAB regularly DAB shouldn't "go off" after freezing unless you have somehow oxidised it during mixing/aliquoting. It would then show as a dark brown aliquot I state this because I still make up my own DAB from dry powder ( have been doing for > 20yrs) Sigma D5637 I dissolve, aliquot and freeze, as you do. An aliquot may be stored at -20C for 2yrs before I defrost/use it If I run out of 50microL aliquots I will defrost a 1ml aliquot, re-aliquot into 50 microL ( when I am doing IHC on a few slides, I make up a small working DAB vol.) Perhaps your H2O2 ( substrate) stock has "gone off"? That will give you a weaker/negative DAB result NB: left-over working DAB solution can be stored at 4C for at least 7 days and still be perfectly usable ( NOT re-usable) If I have only 5-10 slides I will do the DAB step in the humidity chamber, applying DAB as I would antibody (only into the hydrophobic pen-ringed section).

Carl Hobbs FIBMS

Histology and Imaging Manager

Wolfson SPaRC

Guys Campus, London Bridge

Kings College London London SE1 1UL

Answer 2.

I agree with Carl Hobbs: "Perhaps your H2O2 ( substrate) stock has 'gone off'? That will give you a weaker/negative DAB result." If your frozen DAB aliquots keep the same color, I would be very suspect of the Hydrogen Peroxide. It can become unstable very quickly.

Terri L. Braud, HT(ASCP)

HNL Laboratories for Holy Redeemer Hospital

1648 Huntingdon Pike

Meadowbrook, PA 19046