Re: Autoradiography (Quite long reply to 2 questions)

From:"J. A. Kiernan" <>

On Wed, 18 Oct 2000, Cheryl Crowder wrote:

> ... need some help.  A researcher here wants to do autoradiography on 
> mouse tissue.  He wants them paraffin embedded, cut and then the slides 
> given to him for the technique.  I have never done any tissue for 
> autoradiography before.

>   Could someone tell me how thick the sections should be cut (they are 
> tagged with small amount of tritiium)?  Are they put on regular, clean 
> slides or do you use charged or coated slides?

  1. Tritium emits low energy beta particles (electrons), which have
     a trajectory within tissue of about 2 um. This makes it a very
     good isotope for autoradiography because there is very little
     inaccuracy due to particles travelling obliquely into the
     emulsion and causing grains to appear over nearby non-radioactive
     places in the section. 
        The short trajectory also means that if the sections are more 
     than 2 um thick any tritium-labelled  material in the "deeper" parts 
     of the section (more than 2 um from the layer of emulsion) is not 
     detected. For example, in a 10 um section you could have a 5 um
     nucleus labelled with tritiated thymidine in the bottom half of
     the section, next to the glass slide, and its beta emissions would
     never reach the emulsion. Such a nucleus would be falsely negative
     in the autoradiograph. 
        For quantitative work (and all autoradiography should be
     quantitatively assessed) sections for tritium autoradiography
     should be no more than 2 um thick (1 um would be better) if all
     the sources of radioactivity are to be detected. In practice it
     isn't possible to cut large numbers of sections that are regularly
     and reliably all the same thickness, and the variability increases
     as the sections get thinner. Consequently it's more usual to use
     sections much thicker than 2 um and make allowance for the false
     negatives in the course of calculating meaningful results from
     counts of labelled cells, measurements of optical reflectance etc.
     It is still important to have all the sections the same thickness,
     and this should be whatever you are most confident with. For me this
     would be 7 or 10 um for paraffin sections. For a skilled technician
     it might well be  4 or 5 um. Below 4 um there's doubt about what 
     the actual thickness is, whatever the microtome setting. (Probably
     there will be some responding flak against and for this last

  2. The question about the type of slide is a very good one, and I
     haven't seen any published recommendations. Adhesion is important
     not only for the sections but also for the emulsion, which can
     loosen and move during development and subsequent handling. My own
     experiences are limited to plain glass slides, but common sense
     suggests that adhesion of the emulsion (which is a suspension
     of AgBr in aqueous gelatin) should be assisted. The development
     of the autoradiograph (alkaline) is probably the most severe
     challenge to attachment of emulsion and sections to the glass.
        Slides subbed with chrome-gelatin are good for retaining 
     protein-rich sections and other materials in moderately alkaline
     media. Positively charged slides (bought or made in-house with
     APES) are also good. I cannot think of a reason for not using
     subbed or APES slides for autoradiography, and I can't recall
     reading anything about it. A PubMed or Web of Science literature
     search might turn something up. 

  3. This email reply contains numerous unsupported statements. If
     you are interested and need references to respectable sources,
     please ask for more. I'll do my best to provide respectable
     documentation, but it may take a week or two. For exact answers
     to your questions you may need a good library mor than an
     internet listserver.

  Sorry to be so verbose and yet rather discouraging. The best thing
  you could do is to tell your researcher to take a week out and
  read a good autoradiography textbook. The one by the late 
  A. W. Rogers is excellent. 

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1



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