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Problems in Histopathological Technique
Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Email: roy.ellis@imvs.sa.gov.au
A diffuse staining of all tissue structures with Schiff’s reagent following fixation of the tissue with glutaraldehyde containing fixatives.
Glutaraldehyde leaves free aldehyde groups within the tissue after fixation which reacts with Schiff’s reagent to produce a diffuse staining pattern in all tissue structures.
These free radicals need to be removed to produce satisfactory Schiff’s staining. This probably isn’t a problem in a laboratory that doesn‘t do electron microscopy. But it certainly can be in a laboratory that also does EM and where both the histology and EM labs share a common fixative which contains both formalin and glutaraldehyde. The radicals are not easily removed by washing in water.
One Solution!
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One way of removing the radicals is to dewax the section
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rinse in 95% ethanol
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then treat for 1 hour in a mixture of aniline oil and glacial acetic acid. You can use up to 3% acetic acid.
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After treatment the section is rinsed in 95% ethanol
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washed in water then stained in Schiff’s reagent.
Section of kidney stained with PAS after glutaraldehyde fixation and without pre-treatment.
PAS after pre-treatment with aniline oil and glacial acetic acid.
In this second section the Schiff positive material – like basement membranes stand out quite nicely compared to the first section.
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© Roy C. Ellis 2002