Re: [Histonet] RE: pfa vs. formalin

From:Pamela Marcum

At 02:03 PM 8/4/2006, Melissa Gonzalez wrote:
>Hi Jason,
>I was taught by histotechs that 10%Neutral Buffered Formalin is the gold 
>standard vs paraformaldehyde, because it is optimally buffered to exchange 
>with tissue fluids during the fixation process, and that unbuffered 
>fixatives can result in artifacts which you may find microscopically in 
>the tissue slices after stainings. How major/minor this detail turns out 
>overall in the grand scheme of things, I don't really know. I've never 
>seen the direct compare and contrast, for example in H&E sections 
>comparing both fixatives.
>I have found a supplier of 10% Buffered Paraformaldehyde, from Newcomer 
>Supply, which I use routinely for immunofluorescence of perfused, and 
>cryoprotected samples.
>So then I would like to know, is there a technical difference between 10% 
>NBF (formalin) vs 10% NBP (paraformaldehyde)?
>Histonet mailing list


All formaldehyde starts as a powder or flaked paraformaldehyde that is 
convert by heating it in a liquid.  This can be in water or as a 
concentrated solution to be added to a buffer.  Most formulas for PFA are 
2% to 4% or 2 to 4 grams on paraformaldehyde powder dissolved at 60C and 
adjusted with a buffer to the pH as required for the protocol.  It has no 
methanol added to stabilize the formaldehyde solution and is preferred by 
EM and most flow cytometry people.  Some will argue the stabilized solution 
is fine and this really a choice by the laboratory using the solution.

The 10% NBF is made from 37 to 40% formaldehyde solution prepared 
commercially that has been stabilized with methanol (in most cases 10 to 
15%).  It is not a true 10% solution as it is 10mL of the 37 to 40% 
formaldehyde in 90mL water or buffer.  The term 10% NBF is based on the 
volumes used to make the solution and is in reality a 3.7 to 4.0% 
formaldehyde in buffer or water as it is made. It is an excellent fixative 
and is preferred by histologists and pathologist as the majority of papers 
have been written and are based on 10% NBF.

This is an overview I use to explain to students the differences in PFA and 
NBF.  Hope it helps.

Best Regards,

Pamela A Marcum
Manager, Histology Special Procedures
University of Pennsylvania
School of Veterinary Medicine
R.S. Reynolds Jr.  CORL
New Bolton Center
382 West Street Road
Kennett Square, PA 19348

Phone - 610-925-6278
Fax     - 610-925-8120
E-mail - 

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