Re: [Histonet] antigen retrieval question

From:"Bryan Hewlett"


I have often placed sections in hypotonic TBS pH7.6, after the cool down
period, left them for a couple of hours at RT and also overnight, even over
the weekend at 4C, without deleterious effects on any of the markers I have
used. I have heard of people having loss of staining following such delays
however, I have never seen it.
In fact, prolonged (O/N to 48 hour) soaking in hypotonic TBS was a very
common way of unmasking antigens for IHC prior to the days of HIER!
In any case, should such loss of staining occur surely the positive tissue
control would reveal it.
I think that it's worth a test prior to committing your entire set of


P.S. did you get the pictures OK?

----- Original Message -----
From: "Dawson, Glen" 
Sent: Friday, February 18, 2005 9:17 AM
Subject: RE: [Histonet] antigen retrieval question

> Patsy,
> In my experience, staining quality decreases as the time between HIER and
> staining increases.  I believe the epitopes you retrieve with heat do not
> stay open indefinitely and will, in fact, begin to slowly close very
> after HIER.  So, for the best staining, I would recommend no wait time at
> all between retrieval and staining.
> Do you have a tech that comes in earlier than you?  Perhaps they could
> the 3 hr. water bath incubation for you before you get in so you don't
> to wait around.  I have done this myself and it works well if the early
> is available.
> Good Luck,
> Glen Dawson BS, HT & QIHC (ASCP)
> IHC Manager
> Milwaukee, WI
> -----Original Message-----
> From: Patsy Ruegg []
> Sent: Thursday, February 17, 2005 2:33 PM
> To:
> Cc:
> Subject: [Histonet] antigen retrieval question
> I have heard that it is not a good idea to do HIER one day and do the IHC
> the next?  Is this so?  I heard that the unmasking could actually remask
> left sitting a long time before doing the staining.  I have a protocol
> requires a 3 hr. water bath incubation for AR and it would be more
> convienent if I could do that one day and do the rest of the procedure the
> next.
> Patsy
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