[Histonet] Histonet Digest, Vol 269, Issue 6

Sun Apr 12 12:20:50 CDT 2026

I agree, Amos
Empiricism rules, in this context
HIER mechanism is still ???....tho we have figured it out, to great effect!
Maybe, to paraphrase:
"The victorious person in the day of crisis is the person who has the serenity to
accept what they cannot help and the courage to change what must be altered."
However, how do we know what cannot be helped?
Paradigm shifts are always required?
Luverly to read Histonet exchanges again!
Best wishes

Carl

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Sent: 12 April 2026 18:00
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: Histonet Digest, Vol 269, Issue 6

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Today's Topics:

   1. re question regarding pH in retrieval solutions (Gudrun   Lang)
      (Carl Hobbs)
   2. Re: re question regarding pH in retrieval solutions (Gudrun Lang)
   3. question regarding pH in retrieval solutions (Amos Brooks)

----------------------------------------------------------------------

Message: 1
Date: Sat, 11 Apr 2026 18:06:40 +0000
From: Carl Hobbs <carl.hobbs at kcl.ac.uk>
To: histonet <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] re question regarding pH in retrieval solutions
        (Gudrun Lang)
Message-ID:
        <PR3PR03MB6426190D54039DEF85D0D5CCC4262 at PR3PR03MB6426.eurprd03.prod.outlook.com>

Content-Type: text/plain; charset="iso-8859-1"

I hope I set my email to plain text...will find out!
To paraphrase The Excellent Leonard Cohen's song: Nobody knows
?
Chuckle

Citric acid HIER soln is not a buffer if only Citric acid is used
NaOH is used to adjust pH to 6
To make the Citrate a buffer, one would have to use Citric acid and sodium citrate
I use just Citric acid adjusted to pH6 using NaOH
TRIS is, as it is buffered using HCL to pH9
Do any buffers behave as buffers, at such high temperatures?
After Shi, Catoretti was The Man to nail it....imho
Imho, TRIS v Citra HIER rarely work equally well
One or the other  ( or neither ) works on Pwax sections
I have no XP of TRIS v Citra working equally with any antibody
I have not achieved ever, a better result incorporating EDTA into my TRIS pH9
So, for my lab: no "EDTA- sequestering of calcium bound to proteins" is effective
My basic understanding is that high heat induces a dipole moment in many proteins( twisting without fragmentation)  thus epitopes are "revealed", for a time
Need the high heat because the proteins are Formalin-fixed
( if I leave my HIER sections overnight before probing with primary antibody I have a sig loss- of antigenicity)
If I perform HIER on Formalin - fixed cryosections, I can obtain a similar immunoreactivity if I subject sections to 90C HIER....only after an extended HIER time
However, never as high a dilution factor for primary ab
I posted images on the excellent IHC site, several yrs ago....sadly no longer in existence

Well, that is my "rather long in the business" opinion, Gudrun
Discuss?
Best wishes
Carl

------------------------------

Message: 2
Date: Sun, 12 Apr 2026 10:36:02 +0200
From: "Gudrun Lang" <gu.lang at gmx.at>
To: "'Carl Hobbs'" <carl.hobbs at kcl.ac.uk>
Cc: <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] re question regarding pH in retrieval
        solutions
Message-ID: <000301dcca57$66420340$32c609c0$@gmx.at>
Content-Type: text/plain;       charset="utf-8"

Hi Carl,
I am always slightly overhelmed by your answers with my humble school-English ?.
We work with the Roche instruments and detectionsystems. They describe the high-pH AR-solution as "buffer on Tris-basis" and the low-pH AR-solution as "citrate-buffer". So I have to believe, that those reagens are actually buffers. And according to literature pH plays a role in the retrieval-process.
Maybe I need some lessons in chemistry about pH, dissoziation of H+ in buffers and water. Apparently even pure water changes pH with high temperatures and gets slighty acidic.
Boiling in hot water also does the AR-job to a certain degree. Boiling brakes short-range-bonds in proteins and leads to denaturation (and/or koagulation). Denaturation should also play an important part in AR. Boiling causes also the reversal of the binding of methylol-adducts from formaldehyd - and therefore frees the epitopes.
With the calcium-binding-theory I have troubles, because I don't see where the calcium comes from. There are some antigens (like E-Cadherin) that need calcium for stabilization of the tertiary-structure. But are there so many of them?

But why do high-pH and low-pH buffers work different, if the actual working pH is the same?
One idea is, that it is not the boiling-part but the cooling-part. How the proteins fold again during the cool-down would presumably depend on the pH-milieu.

best wishes
Gudrun

-----Urspr?ngliche Nachricht-----
Von: Carl Hobbs via Histonet [mailto:histonet at lists.utsouthwestern.edu]
Gesendet: Samstag, 11. April 2026 20:07
An: histonet
Betreff: [Histonet] re question regarding pH in retrieval solutions (Gudrun Lang)

I hope I set my email to plain text...will find out!
To paraphrase The Excellent Leonard Cohen's song: Nobody knows
?
Chuckle

Citric acid HIER soln is not a buffer if only Citric acid is used
NaOH is used to adjust pH to 6
To make the Citrate a buffer, one would have to use Citric acid and sodium citrate
I use just Citric acid adjusted to pH6 using NaOH
TRIS is, as it is buffered using HCL to pH9
Do any buffers behave as buffers, at such high temperatures?
After Shi, Catoretti was The Man to nail it....imho
Imho, TRIS v Citra HIER rarely work equally well
One or the other  ( or neither ) works on Pwax sections
I have no XP of TRIS v Citra working equally with any antibody
I have not achieved ever, a better result incorporating EDTA into my TRIS pH9
So, for my lab: no "EDTA- sequestering of calcium bound to proteins" is effective
My basic understanding is that high heat induces a dipole moment in many proteins( twisting without fragmentation)  thus epitopes are "revealed", for a time
Need the high heat because the proteins are Formalin-fixed
( if I leave my HIER sections overnight before probing with primary antibody I have a sig loss- of antigenicity)
If I perform HIER on Formalin - fixed cryosections, I can obtain a similar immunoreactivity if I subject sections to 90C HIER....only after an extended HIER time
However, never as high a dilution factor for primary ab
I posted images on the excellent IHC site, several yrs ago....sadly no longer in existence

Well, that is my "rather long in the business" opinion, Gudrun
Discuss?
Best wishes
Carl

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------------------------------

Message: 3
Date: Sun, 12 Apr 2026 12:26:42 -0400
From: Amos Brooks <amosbrooks at gmail.com>
To: "histonet at lists.utsouthwestern.edu"
        <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] question regarding pH in retrieval solutions
Message-ID:
        <CAC95ki8tO3mcNGYcApjDz4EuQ9KfDHaYpi2SDuwsYjcA6VxmVQ at mail.gmail.com>
Content-Type: text/plain; charset="UTF-8"

Hi,
     I always adjust the pH of the solutions at room temperature. What
happens to the pH as the temperature changes should at least be consistent.
So as long as there is a consistent starting point and temperature change
to result in good antigen retrieval, I presume it is a reproducible process
whatever the final pH ends up being. If the end result is consistent then I
have done everything possible to maintain it.

    This is more or less an acceptance of a variable that I can't really
control for. In the words of Karl Paul Reinhold Niebuhr:
"The victorious man in the day of crisis is the man who has the serenity to
accept what he cannot help and the courage to change what must be altered.

All the Best,
Amos Brooks

----------------------------------------------------------------------
> Message: 1
> Date: Sat, 11 Apr 2026 18:18:32 +0200
> From: "Gudrun Lang" <gu.lang at gmx.at>
> To: <histonet at lists.utsouthwestern.edu>
> Subject: [Histonet] question regarding pH in retrieval solutions
> Message-ID: <000301dcc9ce$d82595a0$8870c0e0$@gmx.at>
> Content-Type: text/plain;       charset="iso-8859-1"
>
> Hi all!
>
> Although I am rather long in the business, I came across a question, that I
> wanted to share with you.
>
> With IHC-HIER one uses high-pH tris-buffers at about pH 8-9 and low-pH
> citrate-buffers at pH 6.
>
> Tris-buffers are sensible to changes in temperature ? the higher the
> temperature, the lower the pH.
>
>
>
> A tris-buffer of pH 8,5 at roomtemperatur should have about pH 6,5 at 98?C
> (if my KI-friend calculates correctly).
>
> The citrate-buffer is not affected by temperature.
>
>
>
> So as a result the actual pH in the retrieval-solution would be rather the
> same. That makes me thinking...
>
> Where is the difference? Why do high-pH buffer work mostly better? Am I
> totally wrong with my assumptions?
>
>
>
> I would be happy about any input.
>
> kind regards
>
> Gudrun Lang
>
>

------------------------------

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End of Histonet Digest, Vol 269, Issue 6
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