Sample FAQ Sheet

What kind of tissue samples do I need for SBF-SEM? 

Most are perfusion-fixed tissues perfused with gluraldehyde-based fixative, but some parafomaldehyde-fixed material can be post-fixed. The quality and speed of fixation is paramount.

Can you image cell cutures?

Sometimes – 3D cultures (organotypic) image well, or cells grown on transwell filters (that can be cut).  However monolayer cultures can be tricky because of all the dead space around them when they are embedded, which tends to charge up.

Can I do Immunostaining and see it with SBF-SEM?

Yes – especially of the outer surface of your tissues, using DAB or Gold/silver. You need to limit the detergent , usually.  There are good starting protocols at (References to be added for these).

Are there EM genetic markers that can be used with SBF-SEM?

Yes.  One approach is to use Placental Alkaline Phosphatase, which has long been used for EM, but other methods such as mini-SOG also work. They also work for light microscopy.   (References to be added for these)

I have regular TEM samples in blocks – can I image these using SBF-SEM?

Usually there is insufficient staining to generate a signal  (see here for explanation), but there are exceptions.    You can, however, do either single slice widefield 2D imaging (ie one section, large area, stained as if for TEM, and then montage-imaged at 5nm/pixel) for automated analysis, such as myelin g-ratios.  In addition, you could do serial thin sections onto wafer and stain those (array tomography)

How long does staining take?

Sample staining can take up to a week or more, depending on how thick the samples are.  In addition most people don’t stain every week, but rather in batches .

Should I do staining myself?

Depends on whether you are comfortable working with osmium tetroxide and uranyl acetate and soluble lead salts, and have a fume hood in which you are authorized to do so.   If you have never worked with them before, you should find someone who has done so to help.  Staining itself is not especially difficult, and the variety of recipes indicate that there is a lot of latitude.  Longer duration staining is better, especially the uranyl acetate incubation.   But an major accident with the osmium tetroxide – a volatile heavy metal fixative that can be fatal if inhaled – would certainly spoil your afternoon.