[Histonet] Fixation using Modified Bouins and NBF
John A. Kiernan
jkiernan <@t> uwo.ca
Fri Oct 7 12:02:38 CDT 2005
The modified Bouin that you describe is an illogical
mixture. The purpose of Bouin's fixative is to obtain the
advantages of acetic acid (good nuclear morphology) and
picric acid (rapid coagulation of cytoplasm and strong
staining by anionic dyes) in addition to protein
cross-linking bt formaldehyde.
Your modification uses neutral buffered formaldehyde, which
will oppose the required acidity. The correct recipe for
Bouin's fixative is:
Saturated aqueous picric acid 750 ml
Formalin (37-40% HCHO) 250 ml
Acetic acid (glacial) 50 ml
Fix usually for 24 hours, then transfer to 70% alcohol.
If the tissue is still yellow in the paraffin block, put the
hydrated sections in something weakly alkaline to remove
residual picric acid, then was well with water before
staining. Retained picric acid interferes with some stains,
including those used for blood cells.
If red blood cells are of interest, don't use Bouin's fluid
for fixation because it damages them; use neutral, buffered
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
"Ahmed, T (Tanni)" wrote:
> Composition of Modified Bouins we use is -
> 36gms picric acid
> 200mls glacial acetic acid
> 400mls 10% NBF
> 3.6L Distilled water
> Tissues are fixed for at least 24 hrs. Bouins is changed to 70% IMS
> after the recommended time - i.e 24 hrs however this period can be
> extended up to a maximum of 3 days.
> We are fixing avian bursa specimens in 10% NBF at the moment and
> performing IHC to stain for IBD (Infectious Bursal Disease) using the
> Vector ABC Elite kit. Our pathologist would like to go back to the days
> and try using Modified Bouins to fix bursal tissues then perform the
> same IHC. I have never used toxic MD fixative before and trying to
> convince him that 10% NBF works just as well...I will have a go though!
> no harm in trying old techniques.
> -----Original Message-----
> From: John A. Kiernan [mailto:jkiernan <@t> uwo.ca]
> Sent: 6 October 2005 16:52
> To: Ahmed, T (Tanni)
> Cc: histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] Fixation using Modified Bouins and NBF
> What is the composition of the "modified Bouin's"
> fixative, and how long did the specimens remain in
> it? Back in the old days Bouin's was considered a
> good fixative for some immunofluorescent applications, especially
> localizing protein and peptide hormones. The picric acid in the solution
> subdues much of the autofluorescence.
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London, Canada N6A 5C1
> "Ahmed, T (Tanni)" wrote:
> > Dear Histonetters,
> > I would like to know if fixation of avian bursa tissue with Modified
> > Bouins fixative over 10% Neutral Buffered Formalin has any advantagous
> > effect on immuno precipitation?
> > Thanks in advance,
> > Tanni
> > Tanni S Ahmed
> > Scientific Officer - Histopathology, R&D
> > Intervet UK Ltd.
> > Walton Manor,
> > Walton, Milton Keynes,
> > Buckinghamshire,
> > MK7 7AJ, UK.
> > Tel. +44(0)1908 685552/685543
> > Fax +44(0)1908 685614
> > E-mail: tanni.ahmed <@t> intervet.com
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