In article (Dans l'article)
<01bc8fb7$ce676140$df80808f at bchm7.bch.unp.ac.za>, "brendon"
<priceb at biochem.unp.ac.za> wrote (écrivait) :
>> I have been trying to localise alkaline phosphatase on thawed cryosections
> of blood neutrophils using the method of John Robinson. The basic reaction
> mixutre includes : tricine, TAPS, MgSO4, beta-glycerophosphate, and cerium
> chloride [as the capture metal to make the product precipitate and render
> it electron dense]. So far, no luck whatsoever. The reaction works fine
> in a test tube if I add pure AlkPhosphatase, but not on cryosections. I
> have done a dot-blot on nitrocellulose adsorbed alkphosphatase that was
> fixed by 2% paraformaldehyde, 0.05% glut, pH 7.2 for varying lengths of
> time, up to 1 hour - the activity of pure enzyme is unaffected. Has anyone
> else tried this method, or any other histochemical method that was
> performed on 50 nm sections for TEM??
>> Any input would be greatly appreciated.
>> brendon price
I have not a real solution for your problem, but what I can tell you it is
very difficult to localize an enzyme using its activity on cryosections or
ultrathin sections especially when the cells are fixed and embedded. So
one of the only solution is localized your enzyme using an Ab directed
against an epitope or a part of the Ag or try on ultrathin cryosections
fixed 30 minutes in your fixative and after cryoprotected in saturated
sucrose and freezed in nitrogen vapor
Good luck, Christine