[Histonet] Re: mechanism of staining for Sanderson's with acid fuchsin PMMA embedded bone

From:"Gayle Callis"

Sanderson's bone stain is the same as Stevenels blue developed for PMMA 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.  Continued heating of the solution causes the remaining methylene blue oxidize and the pH continues to increase.  We found our home made Stevenels needed to be topped off frequently, and also filtered since a black ppt  keeps forming.  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. 
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. This paper alone is an education on how dyes work on plastic embedded tissues, including PMMA and GMA.  Horobin RW.  Staining plastic sections:  a review of problems, explanations, and possible solutions.  J Microscopy 131:173-186, 1982.
Some important factors that affect staining of PMMA embedded bone are
Low molecular weight dyes
High temperature 
Alkaline pH 

These factors also affect how hydrophilic EM plastics and other methacrylate embedded tissues stain.  It is well established that toluidine blue, in sodium borate buffer 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.   

Gayle M. Callis

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