Immunomicroscopy: resin techniques and on-section labelling with immunocolloidal gold or immunoperoxidase--planning a protocol.
On-section immunocytochemistry is divided into two parts: (i) processing of biological tissue for section microscopy and (ii) immunolabelling of sections. Many of the more successful microscopical methods employ delicate aldehyde fixation of biological tissue followed by "sympathetic" processing into an acrylic resin. Processing regimens do not have to be complicated. Simple and cost effective room temperature protocols utilising partial dehydration have been devised and they can be as effective as the more complex low temperature techniques in preserving both ultrastructure and antigenic reactivity. The embedded material can be investigated by either light or electron microscopy. Frozen sections can be cut and immunolabelled but only if the tissue is chemically fixed first, as in resin embedding. Fixation with low concentrations of aldehyde will normally better preserve tissue immunoreactivity but this may be at the expense of good ultrastructure with these protocols. If so, low temperature resin embedding methods or rapid freezing and cryosubstitution can be tried. The choice of processing protocol will determine which acrylic resin to use, as will the preference for subsequent immunolabelling with either colloidal gold or peroxidase/diaminobenzidine (DAB). Both types of labelling system offer advantages to localisation studies and can be used in combination for double or even triple labelling. Silver enhancement of the colloidal gold or DAB allows for improved observation by light microscopy.
Hobot
J A
JA
Electron Microscopy Unit, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Newman
G R
GR
D000818:Animals; D005612:Freeze Drying; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron / Q000379:methods*; D051381:Rats; D012116:Resins, Plant; D016610:Tissue Embedding; D016707:Tissue Fixation
D012116:Resins, Plant