TRAP staining decalcified bone paraffin embedded, osteoclasts disappear - (May/14/2018 )

I have been staining mouse femurs that were decalcified in 0.5M EDTA at 4C for 3 weeks using a TRAP protocol from University of Rochester Medical Center TRAP staining paraffin sections.  I see very nice bright red osteoclasts after 30 minutes in the staining solution.  However sometime between this step and mounting, the red stain is totally removed.  I initially rehyrdated in dips of 70, 90, 100, 100 EtOH, then 3x 3min in xylene.  I then tried quick dips in each, including xylene, and it seems to be a little better.  Does xylene remove the fast red violet stain?

Thanks

I note the protocol does not say to dehydrate in ethanol, it just says to air-dry for 15 min, then dip in xylene.

I would follow the protocol exactly as it says, and see if that works.

bob1 on Mon May 14 16:25:49 2018 said:

I note the protocol does not say to dehydrate in ethanol, it just says to air-dry for 15 min, then dip in xylene.

 

I would follow the protocol exactly as it says, and see if that works.

Thank you, I will try this.  In my haste I posted a link to that protocol that is slightly different than the one I have have (also from the same source) that is outdated.  My old protocol from them does say to rehydrate through ethanol, so maybe that's why it was changed.  I will try it and give an update.  Thanks!