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 Ph: 215-938-3689 Fax: 215-938-2021 Honesty AccouNtability AgiLity CoLlaboration CoMpassion -----Original Message----- From: histonet-requ...@lists.utsouthwestern.edu <histonet-requ...@lists.utsouthwestern.edu> Today's Topics: 1. Re: DAB freezing (Alonso Mart?nez Canabal) (Carl Hobbs) ---------------------------------------------------------------------- Message: 1 From: Carl Hobbs <carl.ho...@kcl.ac.uk> Re: [Histonet] DAB freezing (Alonso Mart?nez Canabal) 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) Good luck and...please let us know the answer when you solve your problem Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 ***Please Note: Dec. 1, 2023, Redeemer Health will be updating its email domain from @holyredeemer . com to @redeemerhealth . org. Please alert your IT/cybersecurity team to ensure our new email domain is safe-listed.*** This email, and any files transmitted with it, are confidential and intended solely for the use of the individual or entity to whom they are addressed. The information contained in this transmission may contain privileged and confidential information, including patient information protected by federal and state privacy laws. If you are not the intended recipient, please telephone or email the sender and delete this message and any attachment from your system; you must not copy or disclose the contents of this message or any attachment to any other person. Any views expressed in this message are those of the individual sender, except where the sender specifies and with authority, states them to be the views of Redeemer Health. Redeemer Health may monitor email traffic data. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
