sodium borohydride and autofluorescence

[spiderman]

On Mon, 16 Jun 1997 08:43:19 GMT, a8803349 at unet.univie.ac.at (Martin
Offterdinger) wrote:


>>Actually, in flow we frequently use paraformaldehyde (dilute formalin)
>>but studiously avoid glutaraldehyde.  (EM grade glutaraldehyde is
>>apparently OK and some people use it)  There are some tricks with
>>reducing agents (sodium borohydride) that have been published for flow
>>to reduce flourescece
>What does sodium borohydride do-I mean I know that it is a reducing
>agent, but why should it reduce autofluorescence?? Does anyone know
>this?
>Martin
>
Yes,  fixatives like formaldehyde and glutaraldehyde have the ability
to bind to each other and with cellcomponents without  denaturating
proteins. Some links will produce (aromatic) ringstructures and these
will fluorescence.  These rings are probably produced by  "lose ends"
of the fixative molecules that did not bind to any cellcomponent. By
using sodium borohydride these lose ends will be reduced and thus the
number of fluorescent ring will diminish.

When using sodium borohydride be aware that cellcomponents also will
be reduced. For fluorescence microscopy I used a fixative containing
1% glutaraldehyde, 1% paraformaldehyde and treated my cells after
fixation 2 times 1 minute with 2% sodiumborohydride and rinsed a lott
afterwards. No autofluorescence was detected using a FITC label and of
course a FITC filterblock.

Sebastian