[Histonet] ihc slides

[Goins, Tresa]

Cindy -

I think you got some bad information from Dako tech service.    The buffers used during IHC not only keep the tissue hydrated but provide an environment [salts, pH] that promotes strong binding between the antibody and antigen target.

The antigen-antibody bond is not a covalent chemical bond and will not be maintained during coverslip removal.

So don't go crazy, the slides were shot.


Tresa Goins
Veterinary Diagnostic Lab
Department of Livestock
Bozeman, Montana



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Cynthia Pyse
Sent: Monday, January 24, 2011 5:58 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] ihc slides

Happy Monday Histonetters

I need to pick the brains of the IHC experts out there in histoland. I run
IHC on the Dako autostainer. On one of our runs there was not enough DAB to
dispense on all the slides. My tech dehydrated the slides and cover slipped
them. We printed the stain log on the run and confirmed that the DAB was not
dispensed on three slides. The cover slips were removed and the slide were
soaked in xylene to remove the cover slipping media. The slides were
rehydrated and DAB was applied. There was still no reaction after 10
minutes, The DAB was reapplied for up to 30 minutes with no reaction. Can
anyone out there give me a reason that the slides would not react after the
reapplication of the DAB. I have done this before when the DAB solution was
short on the run and it worked. I can't figure out why this didn't react. I
called tech service at Dako and talked to the supervisor, she agrees with me
that the reapplication should have worked. I checked the run log and the
only missing step is the DAB. The antibodies with the missing DAB have their
own controls on the slides. Any thought out there as to what the problem is.
I have had the PM done on the Dako which should resolve the problem
according to Dako. There has not been a problem since. This mystery will
drive me crazy until I can figure out why. I could use help. Thanks in
advance, I appreciate any input.

Cindy

 

Cindy Pyse, CLT, HT (ASCP)

Laboratory/Histology Supervisor

X-Cell Laboratories

716-250-9235

e-mail cpyse <@t> x-celllab.com

 

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