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
Christopher J. Edwards
Research Associate, Sahenk Lab
Center for Gene Therapy
Wexner Building 2, Room 3224
Lab Phone: 355-5224
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Neuroscience and Cell Biology
Robert Wood Johnson Medical School
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