[Histonet] TRAP Stain Help

[Wait, Trevor Jordan]

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