[Histonet] decal of bone marrows

[Tony Henwood]

Gayle,
A few comments below (in blue):

-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu <mailto:gcallis <@t> montana.edu>
]
Sent: Wednesday, 25 August 2004 8:51 AM
To: Tony Henwood
Subject: RE: [Histonet] decal of bone marrows


Well for one thing, you are going backwards with some potential problems.
Buffered apprx 4.5% formic acid is very gentle for immunostaining, less
destructive to antigen.

I have noticed several postings for "buffered Formic acid" but what is the
pH? and is there a reference for this method? I assume it would need to be
an acidic pH, otherwise Ca2+ would not ionise. Also wouldn't an acetic
acid/sodium acetate buffer at pH 4 or there-abouts work just as well?


If you fix in alcoholic formalin, you create some problems in that acid
cannot ionize calcium in alcoholic conditions. The biopsy needs to
rehydrate from alcoholic solution to decalcify effectively.

The trephines are fixed in alcoholic formalin (ethanol aids the penetration
of formalin into fatty tissues as well as defats the specimen). The biopsies
are then decalcified in AQUEOUS Formic acid/formalin/Sodium Chloride
solution.

You would be better off to fix with NBF totally, then go to buffered formic
acid.  

We tried 10% buffered formalin followed by Formic acid/formalin/Sodium
Chloride solution but the results were not as good as fixing with alcoholic
formalin (better fixation eg clearer nuclear details, well preserved
cytoplasm & granules)

Formic acid with formalin continues to cross link antigens strongly, and the
formic acid here is approx 10% so here you may compromise antigens further
by more exposure unnecessarily to formaldehyde.

But isn't the rule of thumb to have complete (at least adequate) fixation
before the deleterious effects of decalcification begins. Formalin helps to
protect the tissues from subsequent processing. Also HIER is great tool to
apply in these cases.

HCl is a mineral acid and is never buffered.  Formic acid can be buffered
since it is an organic acid. 10% formic acid is generally not buffered
and  made from 90% stock formic, is not bad as it will decalcify faster on
the basis it is more concentrated than buffered formic acid solution.  If
your bone is well fixed, 

(but you said this would be deleterious to immunohistochemical staining!)

you want speed, then 10% formic is not bad, just do not overexpose to higher
conc of any acid.   some people decalcify in
buffered formic overnight (maybe not needle biopsy though).  It is a safer
bet than HCl at any concentration.

If antigen preservation is an issue, then I would probably recommend one of
the EDTA decalcification methods.

Whew, what a lecture - But was it?

Gayle

At 04:08 PM 8/24/2004, you wrote:
>Gayle,
>
>What are the advantages of buffering an acid.
>We use a formic acid/formalin/NaCl mix to decal after fixation in 10%
>formalin/ethanol.
>
>
>Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
>Laboratory Manager
>The Children's Hospital at Westmead,
>Locked Bag 4001, Westmead, 2145, AUSTRALIA.
>Tel: 612 9845 3306
>Fax: 612 9845 3318
>
>
>-----Original Message-----
>From: Gayle Callis [ <mailto:gcallis <@t> montana.edu>
mailto:gcallis <@t> montana.edu]
>Sent: Wednesday, 25 August 2004 12:58 AM
>To: Mildred Fail; Histonet <@t> lists.utsouthwestern.edu
>Subject: Re: [Histonet] decal of bone marrows
>
>
>Question:  Buffered formic acid, commercial preparations?  or do you
>make up an inhouse preparation?
>
>At 07:12 AM 8/24/2004, you wrote:
> >Julie,
> >     We are using Formic acid, with good results on IHC. Rena
> >
> > >>> <Julie.Sanders <@t> med.va.gov> 08/24/04 08:17AM >>>
>
>Gayle Callis
>MT,HT,HTL(ASCP)
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>406 994-6367 (lab with voice mail)
>406 994-4303 (FAX)
>



Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC) 
Laboratory Manager 
The Children's Hospital at Westmead, 
Locked Bag 4001, Westmead, 2145, AUSTRALIA. 
Tel: 612 9845 3306 
Fax: 612 9845 3318 

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