[Histonet] TRAP staining protocol troubleshooting

[Wait, Trevor Jordan]

Hello histonetter heroes!  I have an issue with a TRAP staining protocol that I'm currently using that involves Fast Red Violet LB Salt for the staining of the Osteoclast and then Fast Green as a counterstain.  The osteoclast are suppose to stain Red Violet and the background is suppose to stain green.  However, I'm not seeing any Red Violet Osteoclast but just really really Green tissue.  The bone I was using was cortical bone but surely there would at least 1 Osteoclast in all of sections that I had.  Here is the protocol that I'm using:


Trap Staining solution Mix: Trap Basic Incubation medium, Fast Red Violet LB Salt, and Naphthol AS-MX Phophaget substrate mix


Procedure:

1. Place TRAP staining solution mix in staining dish and pre-warm to 37 degree celsius in waterbath

2. Deparafinnize slides and rehydrate through graded ethanols to distilled water.

3. Place slides in pre-warmed TRAP staining solution mix and incubate at 37 celsius for 30 mins or until control is developed.

4. Rinse in distilled wtaer

5. Counterstain with 0.02% Fast Green for 30 seconds and rinse quickly in distilled water.

6. Dehydrate quickly thorugh graded alcohols, 5 seconds each, clear in Xylene and mount.


The problem is that the main staining with the Fast Red is not very substantial.....at least not as much as it should be.  When I take the slide from the water bath, the tissue section is only slightly red...and may even look more orangish yellow....which is a long ways away from red violet.  Then when I counterstain with Fast Green, I feel that the Fast Green stain is so prominent that it just superimposes the Fast Red stain.  For anyone that has experience with TRAP staining or Fast Red Violet staining then that would be awesome to hear your input!



Trevor Jordan Wait
University of Texas Health Science Center, San Antonio
Class of 2017 MD Candidate
Abilene Christian University Class of 2013 Graduate
B.S.  Biochemistry