Dear Gayle and Interested Histonetters,

The Vector NovaRED peroxidase substrate is extensively tested and quality 
controlled on a variety of tissues, including acetone fixed frozen 
sections, prior to each lot being released for sale. We have never seen 
the substrate cause problems with any tissue morphology. It is somewhat 
difficult to explain the discrepancy you see with other substrates, since 
the buffer conditions (pH, molarity, buffer ions), are the same as for 
AEC for example. The other components of the reagent have no effect on 
morphology, and we have always obtained clean, crisp results on frozen 
and paraffin embedded sections.

What does require further investigation is whether the morphology has 
been affected, or whether it is just the cells are not staining with 
hematoxylin. We've found, in general, that fresher (newer) hematoxylin 
does give weaker staining, and hence incubation times need to be 
increased, or at least adjusted. With time, as it "ripens," the duration 
of hematoxylin staining time should be decreased. 

As a side issue which may be of interest, the Vector NovaRED increases 
sensitivity (signal development) about 2 to 3 fold with incubations times 
up to 30 min, and it will increase slightly after this time with 
incubations up to 1 hour. If, therefore, your staining comes up in 3 to 5 
min with Vector NovaRED, then we would recommend decreasing the primary 
antibody concentration. This will enable you to get the most out of the 
substrate and the primary antibody, and provide crisp, background free 
staining. 

We hope this information may prove helpful.

Sincerely,


Technical Services

Vector Laboratories
30 Ingold Road
Burlingame, CA 94010

Tel:  (650) 697-3600
Fax:  (650) 697-0339
E-mail:  vector@vectorlabs.com
Website:  http://www.vectorlabs.com