Problems in Histopathological Technique


Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011






How to restore nuclear staining to tissue sections which have been exposed to over fixation or over decalcification


The periodic acid method is one (Problem 21) but it doesn’t work on over-decalcified material nor on material treated by fixatives other than formalin.
So if the fixative is Zenker’s:

  • try placing the deparaffinised section into 10% aqueous sodium bicarbonate for 6 to 8 hours - unfortunately most of these fixes are not quick fixes. Then

  • wash the slide in running water for 10 minutes

  • then stain as usual.

Zenkers is a very acid fixative with a pH of 2.3, and Sodium bicarbonate has a neutralising effect on acid material and it may be this neutralisation that allows the dyes to bind to the tissues better with an enhanced staining reaction.


If the fixative is Bouin’s:

  • place the sections into 5% lithium carbonate for 16 hours then

  • Wash in running water for 30 minutes

  • then stain as usual.

Bouin’s contains picric acid and any unbound picric acid left in the tissue will prevent adequate staining. It needs to be removed usually by washing the tissue well in running water before processing. If Picric acid is still not properly removed after the tissue has been processed it can be removed using this method. Lithium carbonate being alkaline neutralises the acidity in the tissue caused by fixation in Bouin’s fluid. But it is really the wash in running water that removes unbound picric acid. In any case it works. As a matter of interest if the formaldehyde in the formula is replaced with formic acid, Bouin's becomes a good decalcifying agent with subsequent excellent staining characteristics in the bony tissue. 24 hours is usually sufficient for small bones and a couple of hours for bone marrow.


If the fixative is unbuffered formalin: you can use 0.05% periodic acid


if the problem is overdecalcification:

  • you can stain in an iron haematoxylin like Weigert’s instead of an alum haematoxylin. Stain in Weigert’s haematoxylin for 30 minutes followed by bluing and differentiation if required. In my experience the differentiation is difficult to control properly with the stained slides looking dirty at the best of times so I would recommend

  • a Celestin blue-Haemalum sequence which gives a much crisper, cleaner result

  • But bear in mind that some overdecalcified material will never stain.

This is a section of bone marrow stained with Weigert's haematoxylin and counterstained with eosin.


This is also a section of bone marrow, from the same block, stained with the Celestin blue - haemalum method and counterstained with eosin.


I know which one I like.


If all else fails using antigen retrieval techniques has recently gained some favour for tissues which have proven difficult to stain.




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© Roy C. Ellis 2002