Help: How to reduce autofluorescence in brain sections?

J. A. Kiernan jkiernan at panther.uwo.ca
Fri Jan 23 11:25:47 EST 1998


In article <Pine.GSO.3.94.980123103211.10068A-100000 at mars.its.yale.edu> Tom Fischer <tmf3 at pantheon.yale.edu> writes:

>Instead of using formaldehyde, try *fresh* paraformaldehyde. The
>formaldehyde off the shelf tends to autofluoresce, as does old (one day or
>more) paraformaldehyde.

  The above statement is is not true. 

>> Does anybody know a method how to reduce the fluorescent
>> background?

  The easiest trick, in my experience, is to use a fluorescent
  label that is excited by green light and emits orange-red.
  Rhodamines and Texas red come in this category. If you must
  use fluorescein, the autofluorescence is less troublesome if
  you excite with near UV rather than blue, with a colourless
  barrier filter. The autofluorescence will then be blue, and
  fluorescein yellow - a bigger spectral difference than when
  the usual filter set for fluorescein is used.

  You can kill all fluorescence in a section by putting the
  hydrated slide in 0.5% osmium tetroxide for 5 minutes, then
  washing for several hours in running tap water. (I can send
  references if you need them.) It is important to remove all
  traces of osmium before doing the fluorescent staining. Of
  course, there is no guarantee that the OsO4 will not oxidize
  something in the epitope that you are trying to detect, but
  that is easy to check with a known positive control.

                                     John A. Kiernan
                                     Department of Anatomy
                                     Univ. of Western Ontario
                                     LONDON, Canada  N6A 5C1

 



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