[Histonet] IHC chromogen loss, HELP!

[Woodward, Denise]

Posting for a friend..........

 

"We have hit a bump in the road with our IHC test and thought maybe you
might have some input as we seem to be stuck.  We stain  (typically for
virus) frozen bovine tissues using Biocare's Mach-3 AP polymer kit and
develop with Vector Red, counterstain, and dehydrate and mount with
Histoclear/VectaMount by hand or xylene/Surgipath mountant on the
autocoverslipper.  We have been noticing a major loss of stain after
development recently. The controls seem to remain stained, it's the more
diffuse and minimally stained sections that we have seen stain disappear
between development and coverslipping. I did a test with Impress-DAB and
Impress-Bajoran Purple to compare and we thought we had solved the
problem as being related to the surgipath mountant. However I just did a
run with cell markers coverslipping with the Histoclear/Vectamount and
there seems to be a loss of stain again. Vector suggested trying 200 mM
TBS for wash/diluent/stop instead of 100 mM. This is a recent problem as
we have been using this protocol successfully for a few years now. Any
suggestions would be greatly appreciated!"

 

Replies directly to Meghan.tucker <@t> ARS.USDA.GOV would be appreciated