Cellular & Molecular Medicine 
Electron Microscopy Facility
UC San Diego
School of Medicine
 


 

This method is used for imaging cell and tissue ultrastructure.


We use a standard protocol that uses a chemical fixation combined with embedding in Durcupan or Embed 812 resin (similar to EPON). These plastic blocks allow sectioning with a consistent thickness, usually between 50 and 80 nm. Using plastic blocks, tissues or cells may be oriented & sectioned a specific way. This method is optimal for obtaining a high degree of morphological & ultrastructural detail and contrast. 


Once the sections have been viewed & recorded, most or all of the information that can be learned from that particular sample has been exhausted. We generally keep the embedded blocks on file in case we need to section them again, but for most projects the work on those blocks will be finished.


Sections from samples embedded in Durcupan and EPON types of resin generally exclude the possibility of immunolabeling. Only densely packed antigens such as hormones may be detectable after etching the plastic sections. There are a few different resins on the market, such as LR White / LR Gold and Lowicryl, that allow antigens to be detected. We do not routinely stock or use these.



For routine transmission EM of plastic-embedded samples for morphology, we can supply a fixation protocol, and we will provide fixative on the day you will fix your samples (we use fresh, EM-grade reagents). If certain samples require a special fixation procedure it is advisable to discuss this with us, and to provide a (published) method beforehand.


Pre-embedding Immunogold & Ultrathin Plastic Sectioning:


Instead of sectioning first and then detecting the antigens accessible on the surface of those sections, the sample is incubated with antibody and gold reagents prior to embedding in plastic & subsequent ultrathin sectioning. The difficulty here is poor penetration of the antibodies and gold, and this method is therefore more suitable for samples where the preparation (such as vesicles or mitochondria) is first isolated from cells or tissues or for whole cells where the target antigens are exposed on the PM. After the immuno procedure further processing and embedding is identical to routine morphology, and the gold is visible within the ultrathin plastic sections.




 

EM morphology image of drosophila brain (courtesy of Jason Goode)

Embedding for Routine Morphology EM