[Histonet] Question on staining

[Jack Ratliff]

Jim,

I would like to respond to your question regarding the pig tibia. After reading Barry's reply, I too question why you would not want to cut thin (5 micron) sections. It then occurred to me that maybe you are not able to do so because this would require the use of a sledge or polycut microtome.

Given the size of your specimen, I agree that you you will have better and more consistent results with your histomorphometry endpoints if you cut thinner sections using a Reicher-Jung Polycut or a Leica SM2500. Upon completion, you can then deplastify the sections (if you use MMA) and yield excellent staining results by employing a Von Kossa reaction with a MacNeals tetrachrome counterstain and/or a standard Goldners trichrome stain.

If you need further assistance or more information, please feel free to contact me

Jack Ratliff

-----Original Message-----
From: Rittman Barry R <Barry.R.Rittman <@t> uth.tmc.edu>
Sent: Wednesday, September 17, 2008 11:39 AM
To: Herrick.James <@t> mayo.edu; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Question on staining

There is a question on the validity of measuring osteoid on thick sections.
 The reason is that it is rare in bone to have sections where edges are absolutely vertical throughout the section. You therefore have several planes superimposed and several edges in these planes resulting in a penumbra effect. The greater the angle to the vertical edge of the bone such as edges of trabeculae or Haversian canals, the greater the errors in measuring.
 The most accurate measurements are those utilizing sections that are 5 microns or below.
 Is there some particular reason that you must use sections this thick?
 Barry

 -----Original Message-----
 From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Herrick, James L.
 Sent: Tuesday, September 16, 2008 5:12 PM
 To: histonet <@t> lists.utsouthwestern.edu
 Subject: [Histonet] Question on staining

 Hi all,

 I am trying to stain for osteoid and bone in pig tibia (need to quantify osteoid and bone volumes). Does anyone have experience with these types of stains in 40-60 µm thick, MMA embedded, sections? I have tried a Masson's trichrome and Toluidine blue, but have been unable to get well defined stain differentiation with this thick of sections. I would really appreciate any help I can get. Thanks again.

 Jim

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