IMMUNOELECTRON MICROSCOPY

• Custom Fixation and Embedding
• Adapting Existing or Developing New Protocols
• Our Standard Methods
• Quantitative Analysis of Gold Labeling

Immunoelectron microscopy protocols include labeling protein targets with antibodies, lectins or enzyme substrates and tagging with colloidal gold which is visible in transmission or scanning electron microscopes. Labeling can be done on fixed fresh tissues, paraffin sections and cultured cell preparations. If you have positive results from western blots or immunofluorescence, the end marker can be replaced with colloidal gold and labeling is done pre-embedding. Pre-embedding procedures are limited to defining extracellular locations unless the cells are permeabilized.

Cells and tissues are often embedded in a resin to facilitate the thin sectioning required for transmission electron microscopy. If the target molecule is intracellular then post-embedding labeling procedures may be successful for epitopes exposed at the section surface. The response of every antigen will be different when subjected to fixation, dehydration and embedding. A variety of fixatives and embedding resins are available in our facility as well as technical expertise and experience.

DEVELOPING NEW PROTOCOLS
If a literature search of the keywords immunoelectron microscopy and your antigen show that no gold-labeling protocols are available for your particular protein, then a new protocol may be developed using the custom fixation, embedding or antigen retrieval protocols described above.

OUR STANDARD STARTING PROTOCOLS
When asked to develop a new labeling protocol, our starting point is fixation in 4% formaldehyde plus 0.2% glutaraldehyde, partial dehydration in ethyl alcohols while lowering the temperature to –20C, and embedding in LR White resin. This protocol has been successful on several sensitive antigens.

QUANTITATIVE ANALYSIS OF GOLD LABELING
Results of gold-labeling experiments are often limited to a qualitative assessment of the gold particle distribution pattern. However, because gold particles can be counted over a given area, the labeling density is often used to compare specimens. The labeling density is sensitive to many protocol alterations and is only a relative measure of epitope in specimens that have been processed simultaneously. Quantitative methods also are available for assessing the clustering of antigen or the coocalization of two antigens. We can teach you these techniques or you can have us analyze the statistical significance of a clustered epitope or the colocalization of two related antigens.