[Histonet] Re: IHC chromogen loss, HELP!

[Sarah Tarran]

Hi Meghan,

We had the same problem with Vector VIP - I stain both atherosclerotic
specimens and burns - for some reason the athero samples were fine but the
burns compeltely lost their colour. It turned out, for us, it was the
alcohol in the dehydration step that was stripping the colour. I would
check the slides after they had been stained and the staining was fine but
once I looked at them after coverslipping there was no chromagen. I now
airdry before taking the slides to histoclear. So my protocol is (from
chromogen onwards):
vector VIP - 15 mins
H20 rinse for 5 mins
Haematoxylin
H20 rinse 5 minutes
airdry (usually overnight but just until slides are completely dry)
histoclear x 2, 5 mins each
vectormount

Hope this helps.

I am not sure with the chromogens you are using but i know you cant use
Xylene with vector blue so this might be the case with the other ones as
well

Good luck


> Message: 10
> Date: Wed, 11 Mar 2009 15:04:07 -0400
> From: "Woodward, Denise" <denise.woodward <@t> uconn.edu>
> Subject: [Histonet] IHC chromogen loss, HELP!
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> 	<40AC6D73C2B95C4CA21B26B7BF380C4002D0E4FF <@t> EXCHANGED.mgmt.ad.uconn.edu>
> Content-Type: text/plain;	charset="us-ascii"
>
> 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
>
>
>
>
>

Sarah Tarran
Postdoctoral Fellow
Vascular Biology Research Centre,
Department of Surgery
Westmead Hospital, Westmead, NSW, 2145
02 98455775
sarah_tarran <@t> wmi.usyd.edu.au