[Histonet] Re: Trichrome stain for MMA and Sandersons Rapid Bone Stain chemical makeup (long reply with protocol)

gayle callis gayle.callis <@t> bresnan.net
Tue Dec 22 14:04:52 CST 2009

Jack Ratiff wrote:

You can do H&E and VVG quite nicely on this type of MMA embedded  
tissue. Maybe you could also try a Sanderson's (methylene blue) with  
the Van Gieson (acid fuchsin w/ picric acid) counterstain???
On Dec 21, 2009, at 11:51 PM, Randall Carpenter  
<rjcarp <http://lists.utsouthwestern.edu/mailman/listinfo/histonet>  <@t>

> wrote:

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > Dear
 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> >
 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > I was
wondering what the best Trichrome stain might be for sawn and  
 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > ground
sections of large stent/artery in methylmethacrylate.  I am  
 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > also looking
to stain elastic fibers.  Any suggestions?  Thanks.
 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> >
 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > Randy
Several years ago  I posted a discussion of Sanderson's bone stain is the
same as Stevenels blue developed for bone work by Maniatopoulos C, Rodriguez
A, Deporter DA, Melcher AH:  An improved method for preparing histological
sections of metallic implants.  Internat J Oral & Maxillofacial Implants
1(1):31, 1987.  The stain was originally developed as a parasite stain.  
Sanderson figured out an easier way to make up the staining solution than
the original recipe, and then it was marketed by Surgipath with trademark.
Her recipe is proprietary and stains with the same results as the
Maniatopoulos method.  We did the comparison in our lab and had identical
results.  Because the Stevenel's is such a royal  pain to make in the lab, I
suggest buying the Sanderson Bone stain.  It is money well spent to avoid a
long day of stain preparation. 
 The chemistry of making this stain is interesting in that potassium
permangante oxidizes methylene blue, forming a thick gooey ppt that takes a
great deal of stirring and heating to get things into solution. The pH is
very alkaline, somewhere in 9 or higher range and when using this stain,
further heating of the solution the methylene blue  will continue to oxidize
and the pH continues to increase.  We found our homemade Stevenels needed to
be topped off frequently, and also filtered since a black ppt  keeps
 Conn's Biological stains Lillie, RD, revised by Stotz EH and Emmel,VM:  H J
Conns Biological Stains. pp 423-424, Ninth ed., Williams and Wilkins Co,
Baltimore MD 1977 has explanation of what happens to the methylene blue when
oxidized by KMNO4.   The by- products of the methylene blue oxidation are
toluidine blue, methylene violet, thionin, Azure A and other Azures  along
with residual methylene blue left in the solution. 
 Some dyes are often found in formulations/recipes for PMMA embedded bone
sections e.g. toluidine blue.    
Staining results are 
Osteoid - blue to intense blue-green; Muscle, connective tissue - blue to
blue-green; Cartilage - blue and/or shades of violet to purple; Calcified
cartilage - medium to dark purple
Calcified bone is stained by acid fuchsin, and light green can also be used.
Basic Fuchsin is also a counterstain


Since PMMA is very hydrophobic, only low molecular weight dyes penetrate the
plastic sufficiently in order to stain all the the described components.
Acid fuchsin also has a low molecular weight. 

RW Horobin has a wonderful publication on the effects of staining on
plastics.  I am not putting my finger on the reference, but this paper alone
is an education on how dyes work on plastic embedded tissues, including PMMA
and GMA.  

Some important factors that affect staining of PMMA embedded bone are

            Low molecular weight dyes

            Temperature - must be heated

            pH - alkaline is important  for good staining.

            These factors also affect how hydrophilic plastics for EM and
other methacrylate embedded tissues stain.  It is well established that
toluidine blue, in sodium borate at pH 11 is a stain of choice for EM
embedded tissues, and requires heating. 

Hopefully this will help in understanding the mechanism. 

I suggest accessing the publications for further information. 

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> Personally,  I
have preferred MacNeals Tetrachrome over Sandersons/Stevenels for
undecalcified bone in MMA, ground sections but the surface must be etched
with 0.7% formic acid and also alcohol etching of MMA, rinsed well, dried
then immersed into the MacNeals.  Do not buy MacNeals as a commercial
preparation, it does not  work as well as an in house preparation.  Here is
the protocol for MacNeals with a basic fuchsin counterstain.  MacNeals can
also be combined with Toluidine blue for a brilliant, well stained bone
section in MMA.     
 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> As in all
staining, young bone takes less time to stain, old bone longer, acid etching
enhances calcified staining by allowing stain to penetrate into a few
micrometers of bone section surface.  This is actually a mild surface
decalcification whereas the alcohol etching softens and probably removes a
very slight amount of  the MMA.  One can always play with staining times,
but do NOT acid etch too long or you will have a section that is too
overstained.   MacNeals has also been combined with Toluidine blue for a
very nice stain combination.  Basic fuchsin on acid etched bone will be very
red, so one cannot be heavy handed with this stain nor van Giesons.  


 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> MACNEAL'S


0.5g methylene blue

0.8g azure a eosinate

0.1g methylene violet

250 ml methanol

250 glycerin

Stir, leave at 50C for 12 hours, then 37C for 3 days.  Filter, store in

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> WORKING

5 mls stock stain solution, 95mls distilled water

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> PROCEDURE:

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> Grind and
polish surface up to l micrometer alumina.  Rinse well with tap water.

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 1.    Etch
surface with 0.7% formic acid for l minute

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 2.    Rinse
with tap water

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 3.   100%
ethanol for 10 minutes, sonicate

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 4.   Stain in
Modified McNeal for 5 minute or longer.  If overstaining with MacNeals
occurs, dilute acid alcohol removes                 stain. 

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 5.   Rinse with
distilled water

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 6.
Differentiate with 70% ethanol with 1 dip

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 7.  0.1% Aq.
Basic Fuchsin 15-30 seconds

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 8.  Brief
distiled water rinse

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> 9.  Air dry, do
not coverslip.  To view, put a white paper under slide, turn light up to
brightest level without filters on microscope,  and view stained surface by
placing a cover slip on top of stained section.  Mounting coverslip will
crack the MMA. 


 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> Gayle Callis 

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> HTL/HT/MT(ASCP)

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