Problems in Histopathological Technique


Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011






To restore good nuclear staining to tissue sections exposed to overfixation in neutral buffered formalin

One of the problems with overexposure to formalin is that over a period of time formalin will bleach tissues. Once the oxidation process has begun in earnest it becomes quite difficult to get sections that are prepared from bleached tissue to stain.


One solution is to rehydrate the section:

  • then treat with 0.05% aqueous periodic acid for 5 minutes.

  • Wash in water for 5 minutes

  • then stain as usual.

Now I don’t know why this works, but it has been hypothesised that as periodic acid is an oxidising agent it may unblock some of the dye binding sites that probably have been blocked by the excess formalin. But whatever the reason it does seem to work providing that the overexposure is not too great. I have tried it on tissue left in acid formalin for three months which were well and truly bleached and which would not stain well using the normal H&E method without pre-treatment then after using periodic acid the results weren’t too bad as you can see from this next slide.



This is a section from a piece of kidney which I purposely left in acid formalin for over three months. It really was quite difficult to stain using a straight forward H&E but after treatment with periodic acid we got this result which I think you will all agree is quite good although it is staining a bit on the red side.




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© Roy C. Ellis 2002