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Published on: February 18, 2016
Immunomicroscopy: resin techniques and on-section labelling with immunocolloidal gold or immunoperoxidase--planning a
1Electron Microscopy Unit, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Insights
Simple room temperature processing effectively preserves tissue for immunocytochemistry, matching complex low-temperature methods. This cost-effective approach maintains ultrastructure and antigenic reactivity for microscopy.
Area of Science:
- Biochemistry
- Microscopy
- Immunology
Background:
- On-section immunocytochemistry involves tissue processing and immunolabelling.
- Successful methods often use aldehyde fixation and acrylic resin embedding.
- Preserving ultrastructure and antigenic reactivity is crucial for microscopy.
Purpose of the Study:
- To present simple, cost-effective room temperature protocols for tissue processing in immunocytochemistry.
- To demonstrate that these methods can be as effective as complex low-temperature techniques.
- To guide the choice of processing protocols and labelling systems for microscopy.
Main Methods:
- Utilizing partial dehydration at room temperature for tissue processing.
- Employing aldehyde fixation with varying concentrations.
- Investigating embedding with acrylic resins for light or electron microscopy.
- Using colloidal gold or peroxidase/diaminobenzidine (DAB) for immunolabelling.
- Applying silver enhancement for improved light microscopy observation.
Main Results:
- Simple room temperature protocols effectively preserve ultrastructure and antigenic reactivity.
- These methods are comparable to complex low-temperature techniques.
- Choice of protocol influences resin selection and labelling system compatibility.
- Silver enhancement improves light microscopic visualization of labelled sections.
Conclusions:
- Cost-effective, simple room temperature processing is a viable alternative for immunocytochemistry.
- Optimized fixation and processing are key to balancing ultrastructure and immunoreactivity.
- Flexible immunolabelling systems (colloidal gold, DAB) and silver enhancement offer versatile localisation studies.
Abstract:
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.
