Stevenels blue

From:Gayle Callis

Stevenels blue stains connective tissue bluish green, osteoid tend to look
a bit washed out but does have the color you described- greenish but can
look very light green.  Van Gieson stains the bone very red, I think it
overstains the bone if you want to see fine structures within bone. VG
should be blotted from bone section rather than rinsed, you can wipe around
section with wet kimwipe or wipe back of slide to remove contaminating VG

There are some interesting things on can do to enhance the bone staining.  

Use formic acid to etch the bone a bit, 0.5% - 1% for 30 seconds, rinse
very well with tap water, blot section completely dry. We used an
ultrasonic cleaner to etch bone. 

Preheat the Stevenels in MW to 56C, do not go over 60C as this continues
methylene blue oxidation by potassium permanganate, a hot waterbath is ok.
After staining temperature is reached, immerse dry section into 56C
Stevenels, let it stand for time you wish keeping in mind there will be
differences in staining due to species, age of animal, type of bone.  DO

Take section out, rinse with hot running tap water, blot dry and view. You
can return the bone for further staining if you wish, increments of 30 sec
to one minute until depth of stain is reached.   

The stained bone is now shades of blue, demonstrating osteoid much better -
your implant should not be stained - but calcification front, Haversian
systems, osteocytes, cartilage, lamellae, caniliculi will all stand out,
very reminiscent of a microradiograph of a ground bone section. Connective
tissue and red blood cells will appear greenish- blue.  

If you counterstain with Van Gieson, beware - the Stevenels is
differentiated out of bone by VG, leaving it less red but more reddish
orange, we opted for no counterstain or use a 0.5% basic fuchsin for

VG in general, stains UNETCHED bone extremely red but you lose the fine
detail of lamellae, all the things described above. 

The pH of Stevenels is approx 9 and as you keep reheating/reusing ( you
should replenish with fresh stain) the pH will continue to increase. We did
this test in the lab, Stevenels is not as stable as one thinks with
continuous heating/reuse. 

A modification of this stain is sold as Sandersons Rapid Bone stain
available from Surgipath, and considering what a pain it is to make up
Stevenels, I would buy the commercially prepared solution - it saves time
with identical results. They also include procedure and counterstains one
can use.   

The original method for bone was Maniatopolos C et al 1987 An improved
method for preparing histological sections of metallic implants Internation
J of Oral and Maxillofacial Implants 1(1):31. 
This publications gives an improved, modified version of Van Giesons,
actually superior to the original VG recipe.  

By comparison of Stevenels and a toluidine blue combined with MacNeal
tetrachrome, the latter combination give far better staining of bone with
implant than Stevenels/VG. The T blue/MacNeals stained osteoid with a depth
of stain not obtained with Stevenels. The MacNeal's also is superior if
made inhouse, commercial solutions were tried and found totally inferior
for undecalcified bone staining.

Good luck

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

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


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