[Histonet] TRAP Stain Help

[Dorothy Hu]

If you include a positive slide to your TRAP? It is a must for each run. It
could be many things if you didn't have positive slide, will be hard to
troubleshot. Also if you see any pink color befor
counterstain/coversliping? If do, there may be because of decolored by
ethanol/xylene and mounting media. You have to use water based mounting
media.
I don't use Sigma kit (tried once), but my protocol is more sensitive. We
do trap on both paraffin and MMA sections. If you need I can forward you
the paper.

Dorothy Hu
HSDM


Message: 2
Date: Mon, 11 May 2015 20:26:14 +0000
From: "Wait, Trevor Jordan" <WaitT at livemail.uthscsa.edu>
To: "Histonet at Lists. Edu" <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] TRAP Stain Help
Message-ID: <1431375976414.44600 at livemail.uthscsa.edu>
Content-Type: text/plain; charset="iso-8859-1"

Hey guys, recently I've used the Sigma Aldrich TRAP Stain Kit in order to
stain for Osteoclasts in Formalin Fixed Paraffin Embedded bone tissues that
are EDTA decalcified.  Unfortunately there was no TRAP stain to be found
whatsoever on all slides that I stained.  I was hoping that someone with
some experience with TRAP stain could really help me out!  Here are a
couple of reasons that I have wondered as to why the TRAP stain might not
be visible....

1. Staining too long with Hematoxylin counterstain?  I have noticed in
several trial runs that sometimes if the Hematoxylin counterstain is too
long then this can effect the amount of TRAP stain that shows up with the
osteoclasts.  Perhaps I'm just over analyzing....

2. Perhaps no osteoclasts were present to even stain....does anyone know
what the usual ratio of Osteoclasts to Osteocytes are?  In the past stains
that I have used....whenever there is an osteoclasts that shows up...it is
usually pretty spotty and many times they are in groups together...is this
how it normally is?

3.  Perhaps the TRAP stain is being washed away through the rinsing with dI
or dehydration with ethanol/clearing just before mounting.


A protocol that I used before the Sigma Aldrich Kit incorporates incubating
the slides in 37 celsius water for 1 hour just prior to allowing the slides
to sit in the TRAP stain solution (this is also 37 celsius in the same
water bath) and letting it sit for 20 minutes.  With this stain...there
seems to be a consistent showing of osteoclasts but I'm just not sure if
all of the osteoclasts are showing up correctly...that is why I moved to
the Sigma Aldrich kit to make sure and the results didn't show ANY
osteoclasts there!  Anyways...I'm just curious if there are any reasons
that the TRAP stain is not showing up...I would appreciate any input!




Trevor Jordan Wait
University of Texas Health Science Center, San Antonio
Class of 2017 MD Candidate