[Histonet] Re: ZINC formalin vs NBF for IHC

[Teri Johnson]

Hi Dolores,

First I need to correct something answered previously. Paula Pierce is absolute correct that zinc will precipitate in the presence of phosphates. You do not want to move them out of Zinc Formalin into regular buffered formalin. However she did say to rinse tissues in PBS or 70% EtOH, but PBS is a phosphate buffer so do not use that. Use regular water, DI or tap, instead. Then place it in alcohol or even regular buffered formalin. Do not put Zinc Formalin on your processor.

Ideally you would want to fix in Zinc Formalin and then start processing in alcohol on your processor. Most places have formalin as their first two stations on the processor so it is difficult to change that in the bigger scheme. When we used this Zinc Formalin in the clinical lab, we would fix for 6-7 hours in Zinc formalin, rinse in water, and then put on the processor with the rest of the tissues and it worked ok.

It does make a difference in antigenicity with some antibodies. But we were never able to completely get rid of antigen retrieval. We just got better staining. You would need to go to a non-formalin type fixative (glycoxal) or use Beckstead's Zinc fixative. That's not something you would want to use routinely but only in special situations. Morphology doesn't look the same and that can cause some issues for some pathologists.

There is a good CD31 antibody out there that works in NBF fixed mouse tissue. Dianova makes a rat anti-mouse, DIA-310 is the catalog number. I don't have one listed for CD34, but I bet someone out there has one.

As for your processes, only you and your pathologist can determine what works best for you. We had no issue using Zinc Formalin in a clinical lab for lymphoma workup and bone marrow specimens. Do not over-fix, it penetrates and hardens tissues more quickly than NBF. Rinse in water and then process using 70% alcohol as your first step if you can. It is possible to successfully integrate this into your lab even if you have to go into NBF on the processor. You will need Zinc formalin fixed control tissues processed the same and will need to re-validate your antibody staining using those. That usually only requires a titration run, which is what you would do with a new antibody lot anyway.

Teri Johnson
Manager, Histology
Genomics Institute for
Novartis Research
Foundation
San Diego, CA
858-332-4752