[Histonet] Toluidine blue staining

[Geoff McAuliffe]

To get consistent staining you must standardize your procedure.

I make 1% aqueous Tol. Blue and 2% aqueous borax (sodium tetraborate) in 
separate containers.
Each day I mix equal parts of each solution so I always use fresh stain. 
My final conc. of stain is 0.5%, doubling to 1% makes no difference.
Stain at consistent temperature in a water bath (60-65 degrees C) for a 
consistent time. Not dark enough? Stain longer and/or at a higher temp.
When I stopped using a drop (how big?) of stain on a hotplate (how hot?) 
until I though it was stained (how long?) I got much better results.
Also, are the biopsy and the mouse tissue fixed and processed identically?

Geoff

Edwards, Chris wrote:
> Hello. Our lab has had highly variable results using toluidine blue to
> stain 1.5 micron thick cross sections of plastic embedded nerve tissue
> from both mouse and human nerve biopsy. It seems like the stain is
> darker in the biopsy tissue than the mouse but both sometimes stain
> equally poorly.  We have tried changing the protocol recipe to make it
> darker by adding more toluidine blue powder but that has not worked
> either. We have used 2 grams toluidine blue, and 2 grams of sodium borax
> with 100 ml of distilled water. We have tried changing the sodium borax.
> Sodium tetraborate-decahydrate and a sodium tertaborate 99.9% metals
> basis from sigma Aldrich have both had the same results. Does anyone
> have any idea how to make it stain darker? I can't really emphasize
> enough how light the blue is. I did read one protocol that involved
> adjusting pH, is that a possible factor? Thanks
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> Christopher J. Edwards
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> Research Associate, Sahenk Lab
>
> Center for Gene Therapy
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> Wexner Building 2, Room 3224
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> Lab Phone: 355-5224
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583 
mcauliff <@t> umdnj.edu
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