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Author Topic: Sequence of events: peroxidase quenching vs. HIER/PIER  (Read 9645 times)

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Offline ImmunoNYC

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #15 on: January 10, 2006, 05:18:40 PM »
Interesting results ... Also it would only be significant if your tissues had endogenous peroxidase to begin with. You should probably consider to include a no peroxidase block control  ... more and more and more slides ;)

Also, I use DAB+ from DAKO for 5 minutes on all of my DAB stains. It really is a superior reagent and I must recommend it!!
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Quote from: "discman"
ok, I finished the stainings today. I tried three different abs (two different anti-CD44, one anti-pan cytokeratin). Although I have to say that the stainings are not yet optimal since I was using a new DAB substrate for the first time (still needs some optimizing), I didn't see any differences between peroxidase quenching and then HIER or the other way around (both before the primary ab).
Maybe there might be some very subtle differences that you only see if you push the detection to the limit, but even staring for quite some time at the slides  :wink: , I didn't realize any differences.

Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #15 on: January 10, 2006, 05:18:40 PM »

Offline broiler25

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #16 on: January 11, 2006, 03:37:11 PM »
If I am using 3% hydrogen peroxide, I know this is somewhat harsh on the tissue, so I use it prior to HIER, therby using it before undoing any formalin crossbridges that are present.  My hypothesis is that the tissue is more stable before HIER than after, so I use it before.

If you are using a weaker peroxidase block (.3 or .03% like many commercially available blocks), it doesn't matter which order you do it.

Offline discman

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #17 on: January 13, 2006, 08:40:51 AM »
Quote from: "MaximinaNYC"
Interesting results ... Also it would only be significant if your tissues had endogenous peroxidase to begin with. You should probably consider to include a no peroxidase block control  ... more and more and more slides ;)

Also, I use DAB+ from DAKO for 5 minutes on all of my DAB stains. It really is a superior reagent and I must recommend it!!
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Hi Maximina,
the tissue does have endo peroxidase (we realized that when a student once skipped the quenching step...). I think I might try the sequence richard was suggesting, sounds interesting. Thanks also for the hint reg the DAB+.

Interesting thought, broiler25, since I didn't find any staining differences, I might do the peroxidase quenching after the HIER in the future.

thanks to all for the interesting discussion and have a nice weekend,
GŁnther

Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #17 on: January 13, 2006, 08:40:51 AM »