Re: [Histonet] toluidine blue staining problem

From:"John A. Kiernan"

You are using a touidine blue recipe for staining
semi-thin plastic-embedded sections, with which
the idea is to stain everything - hence the
alkaline solution of a cationic dye. To use
toluidine blue as a counterstain, you want it to
stain cell nuclei and not much else, and not very
strongly - especially if your silver grains are
over nuclei.

So use the dye at pH 3.5 to 4.0, and rinse in
water adjusted to the same pH. If the staining is
just right, dehydrate in 3 X 100% alcohol. If
staining is too strong, remove some dye with
50-70-% alcohol. Next time, stain at a lower pH.

When I did autoradiography I preferred a red
nuclear stain to a blue one. Brazalum was very
good; you make it up like Mayer's haemalum, but
with brazilin instead of haematoxylin. 
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Stefanie Werner wrote:
> Hello,
> i have a problem with toluidine-blue staining on paraffin sections after
> radioactive in situ hybridization. After developing the slides I stain
> them with 0,2% toluidine blue.
> The procedure is as follows:
>  2 minutes 0,2% toluidine-blue (with or without 1% Borax)
>  rinse in tap water
>  at least 1 minute 30% ethanol (denatured)
>  at least 1 minute 50% ethanol (denatured)
>  at least 1 minute 75% ethanol (denatured)
>  at least 1 minute 95% ethanol (denatured)
>  at least 1 minute 100% ethanol (denatured)
>  at least 1 minute 100% ethanol (denatured)
>  at least 10 minutes Xylol
>  at least 10 minutes Xylol
> The slides are covered using DPX.
> Staining solutions were made in either tap or distilled water.
> After staining, I often see a red background colour on the slide and in
> the tissue under the microscope. Therefore I can't see my
> radioactive signal properly.
> Did anybody have the same problem or has a suggestion to solve the
> problem?
> I also suggested, if the Kodak photo emulsion is the problem, but we
> always use the same emulsion.
> I don't know what to do, because it just appears sometimes, even
> when the procedure is the same.
> Thank you very much in advance.
> Stefanie Werner
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