We dissolve 3,3′-diaminobenzidine tetrahydrochloride in water for immunohistochemistry and the solution becomes dark violet. Is it normal? When dissolving diaminobenzidine (not tetrahydrochloride) with adding of HCl - the solution is light brown, why in our case such result?
Purple is not normal in my experience; our DAB solutions are generally a light yellow-brown. In our laboratory, when we're performing immunohistochemistry, we make our DAB solution (D5905; Sigma-Aldrich) as follows: 1 DAB tablet, 50 mL buffer (we use phosphate buffer, but tris buffer can be used as well if your laboratory prefers it), and 20 µL of hydrogen peroxide (30% stock). On the Sigma-Aldrich website for the product, they specify that the addition of fresh hydrogen peroxide (30% solution) is needed for its reconstitution. That may be the problem, although I don't know for certain as I've never made my DAB solution without hydrogen peroxide in it.
Thank you very much. I think we have a DAB of poor quality (although well-known manufacturer and recently bought). Today we tried to prepare a working solution (bypassing the preparation of stock solution) as advised Christopher - same purple color, and only one hour after adding of hydrogen peroxide color changed to brownish. We used PBS - TBS unlikely to much change the situation. We will buy another DAB. Thanks again for the advices.
I recommend to prepare fresh DAB solutin short time befor you use it. Filter the solution through an 0,2 µm microfilter befor you add the hydrogen peroxid. The colour should be water clear to light brownish. If you like to work with stock solutions you are runnig the danger that the solution would be oxidised by air oxigen. Then the solution becomes brown. The reson for the purple colour could be the wrong pH. The buffers have most the time a pH between 7. 4 and 7.6 (TRIS), destilled water is much more sour.
Thank you very much, Ute. We tried to do directly working solution with PBS, pH is around 7.2-7.4 - close to you mentioned. The same problem - a light purple. Furthermore, we have DAB as a powder and it is itself a dark purple. Previously we used DAB as tablets - they were dark brown. Therefore, we doubt in quality of our DAB.