Re: [Histonet] B5 Fixative - postfixation??

From:John Kiernan

B5 is a near-neutral fixative (pH 5.8-6.0) contrived
to combine the best properties of neutral formaldehyde
and mercuric chloride. 

Formaldehyde penetrates and stops autolysis rapidly, but 
its reactions with proteins are slow. Structural 
stabilization (hardening) takes days. Tissue thoroughly 
fixed (cross-linked) by formaldehyde often shows 
differential shrinkage artifacts - abnormal spaces around 
cells etc - that occur during processing into paraffin. 

Mercuric chloride penetrates the tissue slowly, coagulating
proteins instantaneously as it moves in. The coagulation 
has a finer "grain" than that of proteins coagulated by
alcohol or picric acid, and cytoplasmic organelles 
(secretory granules, and even mitochondria) are preserved
well enough to be seen by light microscopy.

B5 (unlike most mercuric chloride & formaldehyde mixtures)
does not contain acetic acid to coagulate chromatin into 
textures characteristic of particular cell-types. B5 is 
primarily a fixative of cytoplasm that provides crisp and 
informative staining. 

It doesn't make sense to move a specimen from 
phosphate-buffered formaldehyde into a mercuric &
formaldehyde mixture such as B5. If the tissue is 
already fixed (cross-linked) by formaldehyde, later 
reactions of structural proteins with HgCl2 are
unlikely to improve anything structurally. Some 
staining properties might be changed for the better,
but it's easier to treat hydrated slides with
HgCl2 before staining. Treatment with picric acid
has much the same effect, and is commonly done
prior to staining sections of formaldehyde-fixed 
material by trichrome methods.

A specimen fixed in B5 is not going to be improved 
by moving it into an aqueous formaldehyde solution.

 John Kiernan
 London, Canada
An anonymous person (- - )
> Hello all,
> Regarding B5 fixative (not the substitutes), does it have the same effect on nuclear staining after samples has been fixed in 10% NBF and processed?  The question was posed to me during a meeting and honestly, I have never really known.  I know before even fixing, the tissue can be dropped into B5 for a few hours and then switched into 10% NBF to hold until processing.  But for sections that have just been processed routinely and brought to water,  can one place them into B5 and hope to get better than usual nuclear staining??
> Thank you in advance for your assistance.

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