[Histonet] toluidine blue staining problem

From:"Stefanie Werner"


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 

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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