[Histonet] Immuno. confusion

[Troutman, Kenneth A]

Hi Valerie,

The peroxidase blocking step is to prevent endogenous peroxidase activity that will cause a false positive stemming from your application of DAB (the substrate for your DAB solution is hydrogen peroxide).  You can essentially put this blocking step at any point in your stain (following deparaffinization) and before your application of DAB.  I have run this step at varying points in my procedures and they all work fine.  Some antibodies prefer you do this step before primary application and others prior to DAB, but for most stains, it doesn't matter too much.

As for an enhancer, you can make a copper sulfate solution (5mg/mL in buffer).  Or you can try DAB Enhancer or DAB Sparkle from Biocare.

Good luck,

Ashley Troutman BS, HT(ASCP) QIHC
Immunohistochemistry Supervisor
Vanderbilt University Histopathology
1301 Medical Center Drive TVC 4531
Nashville, TN  37232

From: Hannen, Valerie <Valerie.Hannen <@t> parrishmed.com>
Subject: [Histonet] Immuno. confusion
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Date: Wednesday, August 3, 2011, 11:57 AM


Hi folks...

I am going to try to revamp out Immuno. procedure and am looking at a couple of spec. sheets from our primary vendor. I am gettin conflicting information...so I am turning to you all for help.

When doing immuno's that require either HIER or enzyme digestion, do you perform the retrieval first or do you do the "peroxide" step first??
One spec. sheets says retrieve then block endogenous peroxidase...the other says block endogenous peroxidase then do the HIER or enzyme digestion..which is correct??

One other question that I have is...what do you use to enhance your stain?? The enhancing solution that we were using has been discontinued.

Thanks so much!!

Valerie Hannen,MLT(ASCP), HTL,SU(FL)
Histotechnologist
Parrish Medical Center
951 N. Washington Avenue
Titusville, Florida 32796
(321) 268-6111 ext. 7506