RE: Reticulin stain

From:"Monson, Frederick C."

Hi Jason,
    The first time I made up AFS (ammonium ferric sulfate)[(a la, Hdb Chem & Physics, 63rd))] I was told to carefully pick only the lavender crystals (violet in the above book for the compound identified as:  "ammonium iron sulfate(ic)" [NH4Fe(SO4)2.12H2O] and not those that were white.  The latter are identified as "ammonium iron(III) sulfate" [(NH4)2SO4.Fe2(SO4)3].  How the one converts to the other with age in the jar was not explicitly stated and remains unknown to me, but the admonition was VERY clear, if I wanted the reticular or iron hematein stains to work.  While students have departed and lost, it is one area in which I and my mentor have always been in absolute unbending agreement.  [For the reason, that I have never seen any evidence to refute the original admonition.]
If you use only the lavender (violet) crystals, the solution will have a color that will approximate dilute urine, that is, a light, delicate yellow hue. 
As a pure aside, I once retrieved a container in which all the crystals appeared to be white, and even though the white is less soluble in cold water than the lavender(violet) [44:124/L].  I retrieved about 250ml worth of the larger crystals, placed them in a double thickness of #2 Whatman filter paper, and ran a small amount of vigorously boiling water over them to see if I could remove the white and be left with any lavender.  I recovered just enough before they all dissolved.
Finally, yes, I have filtered solutions of AFS(FAS) or whatever, and felt better.  On the other hand, if I make the solution in the morning for an afternoon procedure, the little flecks of stuff are no longer floating and thus not bothersome when I decant from above them.
BTW, when I first tried the Gordon and Sweet as represented by Pearse in 1960(?), I was prewarned, by the author, about the vagaries of the procedure and the variety of 'color' in the result.  Undaunted, I went forward, if only to finally use a method that beautifully counteracted the constant odor of HCHO in the anatomy lab in which I worked.  Gordon and Sweet occupy a lone position at the top of my "most awakening/stimulating" histologic methods.  All the others remain interesting, but only for the results that they provide. 
When I ordered "Ammonium iron sulfate(ic)"(?), I always demanded that it be from a fresh lot and have, on opening, nothing but lavender crystals.  If the lots were fresh, the crystals were as they should have been.
Carlos is correct about the other types of collagen, and you will not be able to speak of "Type III" without doing an adjacent, frozen section with an appropriate Ab.  With respect to Ab's, however, it is still not absolutely clear that an Ab against Type III or Procollagen will 'show' all so-called reticular fibers.  It appears to be an interesting non-conjunction of histology and immunohistochemistry - unless, of course, someone will educate me different!
You are welcome, and come back anytime. 
Hope this helps and gives cheer,
Fred Monson

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail to Geology
West Chester University of Pennsylvania
Schmucker II Science Center, Room SS024
South Church Street and Rosedale Avenue
West Chester, PA, 19383
Phone:  610-738-0437

-----Original Message-----
From: []
Sent: Monday, March 10, 2003 8:36 PM
Subject: Reticulin stain

Hi, I am preparing solutions for a reticulin stain.  This is my first time using Ferric Ammonium Sulfate, or doing a reticulin stain.  When mixing Ferric Ammonium Sulfate in Distilled Water is the solution supposed to be Rust/Light orange colored with a white precipitate that will not dissolve?  If so is this solution supposed to be filtered?  Any other recomendations would be appreciated, thanks for your time.

Jason Sullivan
Chicago, IL

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