Re: paraformaldehyde and colloidal iron staining of mouse tissue

From:Geoff McAuliffe

HI Sharron:

Sharron Ladd wrote:

> Dear Histonet,
> I  work in a University research setting. I have been helping someone
> who knows absolutely nothing about histotechnique with Colloidal iron
> staining of various mouse tissues (including liver, spleen and kidney).
> The mice were perfused with 4% formaldehyde (made from paraformaldehyde)
> and the tissues were then stored in 10% neutral buffered formalin (made
> in the lab). He is using Van Gieson solution as a counterstain. He tried
> the stain several times and each time his sections turned green.

If the van Gieson counterstain is turning the sections green, don' t use
that counterstain.

> So, we
> each ran the stain side by side and threw in human appendix that had
> been fixed in commercially made 10% neutral buffered formalin.
> Everything turned green except the appendix!!

This is not a good control because it is a different kind of tissue AND a
different fixative. You must run the same tissues (divided in half) exactly
the same way changing only the fix.

> I have read many of the past postings about paraformaldehyde vs.
> formaldehyde, especially Dr. Kiernan's postings. Many, many people here
> at the university make their own 4% formaldehyde (from paraformaldehyde)
> and that is all they will use. Dr. Kiernan said in his posting that the
> reason this is done is "tradition, dating from about 1960." Some
> professors claim morphology is better. I also think it is used because
> some of the tissues used to be set aside for EM and 4% formaldehyde
> (from para) worked for both EM and light microscopy. I have 10% formalin
> on my tissue processor only for running cell blocks, but some people get
> very upset when they see it there. They don't want 10% formalin touching
> their tissue.
> In the colloidal iron staining situation above, I suggested perfusing
> the animals with 10% NBF instead. I got the same reaction as always,
> WHY?--they are both formaldehyde and what if the methanol in the
> commercial preps does something awful to my tissue??

The methanol is probably doing very little, if anything, to your tissue.

> This email is getting awfully long, so I guess what I want to know
> is...Can perfusion fixation in 4% formaldehyde (made from
> paraformaldehyde)  interfere with colloidal iron staining?

Not in my experience (with kidney).

> Secondly, how
> can I convince people that  there is no reason to fear commercial
> preparations of formaldehyde (especially since no one in my department
> does EM anymore)?

You may not be able to. Old habits die hard, if at all.
On the other hand, if you make your own fixaitve from either
paraformaldehyde or commercial formaldehyde (the 37%-40% stuff from Fisher
Scientific) you will always know what is in your fixative. You won't have to
worry about the manufacturer changing the formula or the vendor substituting
something that costs him less.

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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