Aidan,

Never done it, but I'm pretty sure that HRP would be dead and buried by the
time you hit the counterstain, let alone dehydrated, coverslipped,
decoverslipped (is this a word?) rehydrated etc.

It's my understanding that 7-10 minutes exposure to H2O2 will kill
peroxidase activity, so the action of putting on your substrate signs its
death warrant.  You can extend the life of your HRP to half an hour or so,
by generating small amounts of H2O2 in situ using a glucose oxidase-glucose
adjunct to your subtrate, instead of H2O2.  Don't forget as well that the
HRP is probably buried as well under a pile of polymerized DAB, so were it
still active your substrate would have to get past this barrier too.

You might be able to reincubate with secondary and/or tertiary (assuming
some of your primary and/or secondary is still bound.  What I would do in
your situation is remove the coverslip, rehydrate and repeat the whole
procedure...or even better, start again on a new section

Simon 

Simon Smith B.Sc. AIBMS
Supervisor, Laboratory Resources
Skeletech, Inc.
22002 26th Ave SE, Suite 104
Bothell   WA   98021
Voice: (425) 424 2663   Fax:  (425) 424 2600
E-mail: ssmith@skeletech.com  


-----Original Message-----
From: Pam Marcum [mailto:pmarcum@polysciences.com]
Sent: Wednesday, May 03, 2000 6:18 AM
To: a i d a n s c h u r r; histonet@pathology.swmed.edu
Subject: RE: re-DAB-in slides


It has been awhile but I have had the to remove coverslips from HRP stained
slides and it fades only slightly.  I did make sure it was a limited
exposure to xylene and watched for the coverslip to come loose.  Pam Marcum

-----Original Message-----
From: a i d a n s c h u r r [mailto:Aidan.Schurr@hvh.co.nz]
Sent: Tuesday, May 02, 2000 6:51 PM
To: histonet@pathology.swmed.edu
Subject: re-DAB-ing slides


Hmmmm...

Does anyone know if the HRP survives the dehydrating process
when coverslipping?  ie. is it possible to de-coverslip an
'underdeveloped' slide, and re-incubate in DAB?  note: we have a
film coverslipper, so would be de-coverslipping in acetone - this
may be a negative factor!?

Thanks,
Aidan

___________________________________________________
a i d a n   c   s c h u r r
     mlso,  histology department
      hutt valley health
       lower hutt, new zealand

     ph.  ++64 4 5709173
     fax  ++64 4 5709214
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