problem with autofluorescence caused by glutaraldehye

[Steve Rogers]

In article <hfang.760213931 at sfu.ca>, hfang at fraser.sfu.ca (Hung Fang) wrote:
> 
> 
> Hi, there!
> 
>         Does anyone out there know any methods to reduce or eliminate the
> autofluorescence caused by glutaraldehyde fixation?  Somebody has suggested
> that treatment with sodium borohydride (1mg/ml) might work. However, it has
> not worked in my hand so far. Has anyone ever used sodium borohydride, and
> has a successful experience? 
> 
>         BTW, I am working on fixed entact sea urchin embryos, not sections, 
> and I don't know whether this might make any difference. Also, the autofuorse-
> cence caused by glutaraldehyde fixation seems to have a quite broad spectrum,
> therefore, the problem can not be solved by chosing another kind of exitation
> filter.  Oh, also, for some experimental reasons, I could not use fixatives 
> such as formaldehyde, methanol.
> 
>         Any suggestions are appreciated!
> 
> 
>     
I routinely use glutaraldehyde on cultured cell lines.  It is crucial that
you use the lowest concentration of fixative you can get away with, without
loss of ultrastructural preservation.  I typically use 0.3-0.4%, but I have
seen protocols use up to 1% successfully.  I also use NaBH4 to quench
unreacted aldehydes.  Three fifteen minute washes with 1mg/mL NaBH4 in PBS
(pH 8) works for me.  The higher pH decreases the rate of borohydride
degradation.  

I've only used this technique on thin (4-10 micron) cultures, so my
suggestions may not be suitable for whole embryos.  What are you looking
at?