[Histonet] TRAP Stain Troubleshooting

[Wait, Trevor Jordan]

Hello Histonetters!  I have a few questions regarding TRAP Staining and I'm hoping someone who has experience with this stain could help me out.  I have been using a protocol that is apparently not goo because it hasn't shown any TRAP whatsoever.  I have seen various themes in several sources, one of those is that slides are incubated for 1 hour in pH 9.4 TBS before being put into the TRAP staining solution.  My question for this is why?  In my protocol, I have a TRAP incubation medium that contains the tartaric acid, that is at pH of 5.0, which I then combine with Naphthol and Fast Red (which are the reagents responsible for the chromogenicity of the stain).   I guess I'm confused as to what the tartaric acid is used for as well....which I'm speculating that it denatures most of the enzymes other than TRAP (hence the name Tartrate Resistant Acid Phosphatase).  Therefore, I see the three main steps for this protocol to be:


1. Incubate in TBS pH 9.4 (which I'm still confused why)

2. Combine with Tartaric Acid to denature all of the other enzymes to localize osteoclasts

3. Add Naphthol and Fast Red to Identify where TRAP is.


Therefore, I'm confused why I am combining Tartaric Acid with my Napthol and Fast Red....is this a common method that is done to eliminate multiple unnecessary steps?  If somebody has a protocol that works consistently for them and wouldn't mind sharing that with me, I would very appreciative of you!  I'm just trying to understand this stain so that I can somehow tweak it to work.


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