Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Background in immunostained cartilage
I have tried to immunostain sections of whole mouse embryos with several primary antibodies to a nuclear epitope. I am getting nonspecific antibody staining in cytoplasm and in the connective tissue around the cartilage.
I have blocked with embryo powder, normal goat serum, normal horse serum, beat blocking solution from Zymed, and Fab fragments. What could be reacting with secondary alone?
I do a lot of cartilage and bone IHC markers, mostly on rat, but have done some mouse tissue. Is your primary made in a mouse? Even with rat tissue, anti-mouse secondaries can combine non-specifically with the rat tissue, I put rat serum in my detection and it helps tremendously with the background.
The different blocking steps you have tested all block hydrophobic areas ("sticky sites") in your specimen. Hydrophobic areas are blocked before the immunoincubation with e.g. normal serum or BSA. Once blocked these sites generally will not give rise to background anymore.
Cartilage and perichondrium are composed of collagen fibers with a positive charge (still present after aldehyde fixation) embedded in proteoglycans which have a negative charge. Most antibodies (primaries and secondaries) are negatively charged at pH 7-8.2. I therfore think that the collagen fibers present in the cartilage tissue are causing your background problem. This charge-determined background can be circumvented by adding negatively charged molecules (e.g. aurion BSA-c) to the wash and incubation buffers. Another possible cause for background (a specific binding to proteoglycans) can be prevented by adding gelatin to your buffers. Do not put both BSA-c and gelatin in the same buffer, because they have charge-determined affinity for each other as well.
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Peter van de Plas