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Author Topic: Sequence of events: peroxidase quenching vs. HIER/PIER  (Read 9919 times)

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Offline discman

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Sequence of events: peroxidase quenching vs. HIER/PIER
« on: January 09, 2006, 02:48:01 AM »
Hi all,

might be a stupid question, but I found various protocols that use different 'sequences of events'. What do you do first? Antigen retrieval beforeperoxidase quenching, or the other way around? Is there a rationale for one or the other, or doesn't it really matter?

Thanks in advance,
Günther

Sequence of events: peroxidase quenching vs. HIER/PIER
« on: January 09, 2006, 02:48:01 AM »

Offline n_bjorkhammer

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Protocols for HIER and Peroxidase
« Reply #1 on: January 09, 2006, 08:30:09 AM »
Hello,

I have not seen any difference in immunostain intensity between using the peroxidase blocking prior to HIER, or any other pre-treatment, in comparision to applying pre-treatment before peroxidase blocking.

Our protocol suggest doing the blocking before pretreatment, but no reference to why...

Nick

On another matter... Can anyone clarify if peroxidase blocking is still needed when using the "new" polymer based detection system?
Niclas Björkhammer, MSc
Technologist II - anatomical Pathology and Morgue
Department of Pathology
Sidra Medical and Research Center
Doha, Qatar

Offline excalibur

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #2 on: January 09, 2006, 10:23:32 AM »
No, peroxidase blocking is not needed for the polymer or alkaline phosphatase based systems.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline ImmunoNYC

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #3 on: January 09, 2006, 10:35:09 AM »
Not true, peroxidase blocking *is* necessary, at least with the polymer kits I have used as they are still HRP based and you develop with DAB.

Most of them come with their own blocker though so it takes a mere 5-10 minutes. I find in some cases I still need to use my own peroxidase block especially if working with bone marrow or spleen etc.
[/color]

Quote from: "excalibur"
No, peroxidase blocking is not needed for the polymer or alkaline phosphatase based systems.

Offline ImmunoNYC

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Re: Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #4 on: January 09, 2006, 10:37:45 AM »
I do peroxidase blocking after antigen retrieval. In fact I do all blocking (protein, biotin etc) after antigen retrieval as I am concerned antigen retrieval may enhance "background". This is definitely a concern when biotin blocking, but for peroxidase it's more superstition than experience.[/color]

Quote from: "discman"
Hi all,

might be a stupid question, but I found various protocols that use different 'sequences of events'. What do you do first? Antigen retrieval beforeperoxidase quenching, or the other way around? Is there a rationale for one or the other, or doesn't it really matter?

Thanks in advance,
Günther

Offline excalibur

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #5 on: January 09, 2006, 10:42:33 AM »
I don't block peroxidase when using the Dako Envision kit.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline discman

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #6 on: January 09, 2006, 11:00:23 AM »
Following my gut feeling, blocking after antigen retrieval would seem right and that's what I usually do... Anyway, I tried both versions today and I'll see tomorrow if it makes a difference (at least with the ab I'm using today... ;-) )

Thanks for your replies.

Günther

Offline ImmunoNYC

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #7 on: January 09, 2006, 01:31:30 PM »
I suppose then that that means your tissue does not have endogenous peroxidase? Why wouldn't you use the block provided with the kit? There is nothing in this kit that magically makes endogenous peroxidases disappear.

Quote from: "excalibur"
I don't block peroxidase when using the Dako Envision kit.

Offline ImmunoNYC

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #8 on: January 09, 2006, 01:32:02 PM »
I am curious to hear your results!![/color]

Quote from: "discman"
Following my gut feeling, blocking after antigen retrieval would seem right and that's what I usually do... Anyway, I tried both versions today and I'll see tomorrow if it makes a difference (at least with the ab I'm using today... ;-) )

Thanks for your replies.

Günther

Offline excalibur

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #9 on: January 09, 2006, 02:38:34 PM »
Hello Maximina,

the Envision G/2 AP kit does not come with block and the protocol does not even list a hydrogen peroxide step, just as alkaline phosphotase detection kits do not need the H2O2 step. It also eliminates the need to block endogenous avidin and biotin. I think it would be an ideal sytem for you to try, especially on bone marrows and other bloody specimens.

Biocare also makes a similar kit they named Mach 3.

Unfortunately, the kits are currently only for human tissues as they are a cocktail for mouse and rabbit antibodies. You may be able to persuade them to produce kits for animal tissues.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline richard03

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #10 on: January 09, 2006, 09:13:33 PM »
Blocking endogenous perxidase after AR may be safe since you don't know what is going to happen after AR (endogenous biotin often revealed after AR).

I have indeed found some differences for H2O2 blocking before and after primary antibody incubation. Blocking before primary antibody incubation produces slightly weaker staining than that after primary antibody incubation. So I always block endogenous peroxidase after primary antibody just to be safe.

Richard

Offline ImmunoNYC

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #11 on: January 09, 2006, 11:05:21 PM »
Oh!! Of course if you are using an AP kit you do not need to block. That you were using AP wasn't clear to me before.

For my tissues I use EnVision or EnVision Plus Peroxidase kits and hence OF COURSE I need to block.

I am a DAB fanatic so am not willing to change my stubborn ways. However, I recently am having an affair with AEC but I feel it will be tempestuous and ultimately I will go back to DAB who is my main man.

[/color]

Quote from: "excalibur"
Hello Maximina,

the Envision G/2 AP kit does not come with block and the protocol does not even list a hydrogen peroxide step, just as alkaline phosphotase detection kits do not need the H2O2 step. It also eliminates the need to block endogenous avidin and biotin. I think it would be an ideal sytem for you to try, especially on bone marrows and other bloody specimens.

Biocare also makes a similar kit they named Mach 3.

Unfortunately, the kits are currently only for human tissues as they are a cocktail for mouse and rabbit antibodies. You may be able to persuade them to produce kits for animal tissues.

Offline ImmunoNYC

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #12 on: January 09, 2006, 11:06:17 PM »
Fascinating observation Richard, I will try this myself on some of my "weaker" stains.

Quote from: "richard03"

I have indeed found some differences for H2O2 blocking before and after primary antibody incubation. Blocking before primary antibody incubation produces slightly weaker staining than that after primary antibody incubation. So I always block endogenous peroxidase after primary antibody just to be safe.

Richard

Offline discman

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #13 on: January 10, 2006, 11:21:28 AM »
Quote from: "MaximinaNYC"
I am curious to hear your results!![/color]


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.

Günther

Offline discman

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Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #14 on: January 10, 2006, 11:23:38 AM »
Quote from: "richard03"
I have indeed found some differences for H2O2 blocking before and after primary antibody incubation. Blocking before primary antibody incubation produces slightly weaker staining than that after primary antibody incubation. So I always block endogenous peroxidase after primary antibody just to be safe.

Richard


wow, this is interesting. I never thought about quenching after the primary! I guess this means that there are more things to try out there...

Sequence of events: peroxidase quenching vs. HIER/PIER
« Reply #14 on: January 10, 2006, 11:23:38 AM »