[Histonet] toluidine blue for cartilage with controls and mast cell staining

Gayle Callis gayle.callis at bresnan.net
Tue Jun 2 17:38:50 CDT 2015

You wrote:  


We use a canine mast cell tumor as positive control - veterinary lab

Probably looking for mast cells in the core.




-----Original Message-----

From: Bernice Frederick [mailto:b-frederick at northwestern.edu
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> ] 

Sent: Tuesday, June 02, 2015 12:02 PM

To: Histonet at lists.utsouthwestern.edu

Subject: [Histonet] Toluidine blue


Hello all,

I was taught to do Toluidine Blue O without a control. Is there actual one
and what would it be? I'm staining a bone core. Don's ask why, it's research
and what a researcher wants... Plus they have a protocol they are following
for this cartilaganous defect.




Bernice Frederick HTL (ASCP)

Senior Research Tech

Pathology Core Facility

Robert. H. Lurie Cancer Center

Northwestern University

710 N Fairbanks Court

Olson 8-421

Chicago,IL 60611



Bernice and Tresa, 


Having done a bone research study like this in the past, controls should be
and were carefully done.  You need to know normal cartilage from treated or
defect in cartilage.  The researcher certainly should have set their
experiment up accordingly but may have controls in place now???    You did
not say if this is articular cartilage from exterior joint surfaces where
they took the core or deeper in the bone at the growth plate?  These two
cartilages will stain differently with T blue.  Normally and when studying
articular cartilage defects, it is wise to also do a safranin O/fast green
stain along with the T Blue.     Controls are extremely important and need
to be carefully set up.   Hopefully, you have a contralateral bone normal
core from the same animal OR a core sample from an untreated, naive control
animal.    It was never stated what the experimental animal model is being
used?     I have done a study like this in the past.    


When core is  decalcified with an acid or EDTA, then the control needs to be
decalcified exactly the same way and at the same time as experimental cores
with defect.  If you are decalcifying with EDTA, then you should have a
normal core that is not decalcified.  This is difficult with mouse but
possible with larger animals.  The reason is to see if the proteoglycans in
the articular or even the growth plate cartilage will be extracted by EDTA,
and not appreciably by buffered formic acid.  Articular cartilage where
proteoglycans have been removed by a decalcifying agent will have different
tinctorial quality (lighter) than cartilage never exposed to a decalcifying
agent.  EDTA is used by biochemists to extract proteoglycans for biochemical
studies, and will the same thing in a cartilage section.  Hence, there will
be less staining seen with the toluidine blue or the Safranin O/fast green
stain after EDTA.  Hence you should run two controls, 1)a decalcified
cartilage control and 2) an undecalcified control.   How you decalcify will
be important in order to retain proteoglycans in the cartilage.  I strongly
suggest using buffered formic acid, available commercially.   You will find
recipes for buffered formic acid in text books that contain sodium formate
or sodium citrate.  Look for these ingredients in product MSDS before you
buy the formic acid decalcifying solutions.   If there is any question about
EDTA versus buffered formic acid and other acid decalcifiers i.e HCl, Nitric
acid, etc.  for cartilage studies, I will be happy to send publications
concerning this topic privately.   


The Toluidine blue that we do for cartilage is designed to show cartilage
staining and not mast cells.  It could be the mast cells might be seen along
with the cartilage staining but that is not the point.     


The toluidine blue stain I do for mast cells is entirely different from the
toluidine blue cartilage staining protocol.     


I will be happy to send you a toluidine blue stain procedure for cartilage
and also the SafO/Fast green protocol.   I have a superb  T blue mast cell
stain from Churukian which allows mast cells to stand out without any blue
background in surrounding tissues that is often seen with other T blue
staining protocols.  


Hope this helps.  


Gayle M. Callis




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