Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Paraffin or frozen sections for immunohistochemistry
Are paraffin or frozen (fixed) sections are better for IHC? I've had great success in the past with frozen or vibrating microtome sections, and have been trying paraffin lately, but haven't got any good results.
Generally frozen sections are better for IHC because the antigenic content is well preserved (provided the tissue is snap frozen rapidly, preferably in isopentane, then stored at -70C). A "good" frozen section cut at about 5 microns should provide adequate morphology.
The advantages of paraffin tissue blocks is that larger pieces of tissue can be used, and morphology is a degree better, storage is easier, etc.
The disadvantage of paraffin blocks is the fact that the processing of the tissue (especially when preserved in common fixatives such as formalin or other formaldehyde-based solutions) cross-links certain proteins in and on the cells. Preatreatment to "unmask" cross-linked antigens is often essential. Antigen retrieval techniques include microwaving in citrate buffer and pressure cooker techniques. However, some antigens are destroyed by paraffin processing, so for these the manufacturer of the antibody should recommend the use of frozen sections only.
Cambridge Antibody Technology
The Science Park, Melbourn,
Royston, Cambridgeshire SG8 6JJ
In general, immunoreactivity is often better in cryostat sections than in wax sections, however tissue morphology is usually not as clear. If you are getting satisfactory results with cryostat sections, then I would probably recommend sticking with that technique. However, if need to use wax sections for whatever reason, there are several ways of tweaking the protocol to try and improve the staining. Any good IHC text book will outline most of these.
Off the top of my head, I would suggest playing around with the fixation conditions or trying some form of antigen unmasking
step (particularly if you are currently seeing no specific staining at all).
Ian Jones, PhD
School of Biological Sciences,
Queen Mary and Westfield College,
University of London, England.