toluidine blue stain for bone and cartilage

From:Gayle Callis

I always found T blue with borate too blue for bone and cartilage UNLESS it
is used for EM/epoxy resin work (Smuckler, Benditt et al).   We use either
a 1% toluidine blue for 1 min at RT, aqueous, no buffering, rinsed well
with water, air dried and coverslipped to avoid any removal of T blue by
alcohols.  If you end up with too blue a color, dilute the stain to 0.1%,
control time and air dry.  

One of the best methods was published in J of Histotechnology, Eurell and
Sterchi, Microwaveble toluidine blue stain for surface staining of
undecalcified bone sections.  17(4):357-359, 1994.

DON'T LET THIS TITLE FOOL YOU! This method also works on decalcifed bone
sections, and uses a phosphate buffer at pH 8. The stain is warmed in a
microwave to stain PMMA sections and you can do decalcified bone with
cartilage at RT, up to overnight.   

One important thing about T blue staining on cartilage is to remember what
the age of the animal is, plus species, and cartilage, also if EDTA was
used to decalcify, that will remove proteoglycans and change tintoral
quality of t blue staining. 


>Does anyone have the procedure for the Toluidine Blue stain with sodium
>borate.  We routinely use this stain for articular cartilage, our current
>protocol uses a sodium acetate/ acetic acid buffer, but I'm always willing
>to try something different.
>Liz Chlipala
>BolderPATH, Inc.
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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