[Histonet] RE: DAB: old-fashioned style

Hobbs, Carl carl.hobbs <@t> kcl.ac.uk
Mon Sep 1 14:05:35 CDT 2008


I do it in the Old-fashioned way too;-)
May not be as sexy but, works every time and the costs are small, relative to commercial kits...;-)
I jest prefer to make a bulk and freeze the aliquots, rather than weigh out DAB!
So: 5g  of Tim's most excellent "dust"...aka DAB.
Place 50 1-5ml m'centrifuge tubes in a rack.
Heat 50ml distilled water in a 250ml beaker to  ~30C
Add enough heated water to the 5g bottle to 3/4 fill it, screw on the lid, mix gently,  in a fume cabinet.
Return this to the 250ml beaker. No more than a couple of backwards and forwards between bottle and beaker will ensure complete dissolution of stock DAB.( yep, if you wish , add a magnetic stirrer bar)
Aliquot 1ml into each, in a rack.
Place rack in freezer for a couple of hrs while you have lunch......store in a box at -20C

I like to incubate my slides in a 200ml container of DAB ( I do not usually like to apply it sequentially onto slides on a tray....such a pain but, but there are  always exceptions. So you can make up smaller aliquots, if you wish! Or just use 200mls...the cost is trivial).
I defrost a 1ml aliquot of DAB in running hot water, make up my 200ml TB buffer ( from a 10x stock), add 200microL 100vol H2O2, my 1ml of defrosted DAB stock, mix well and add my rack of slides, gently up-and-down the rack a couple of times and then leave for 10 mins.
Then take out rack into tap water and give it a good rinse in running water before going into Haemalum.....etc
Carl
PS:  do not leave the DAB aliquot in hot water for longer than it takes to dissolve/mix all solutions well.
Also, reaction of H2O2/HRP/DAB should go to completion within 5mins but....after  10mins, imho, using above relative volumes,  the effective reaction is complete, for my IHC reactions.
Sure, I am very happy to state my ways, if only to test them within Histonet!
Constructive critiscisms would be very welcome.



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Subject: Histonet Digest, Vol 58, Issue 1

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Today's Topics:

   1. RE: (para)formaldehyde (Tony Henwood)
   2. RE: (para)formaldehyde (Tony Henwood)
   3. RE: DAB: old-fashioned style! (Tony Henwood)


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

Message: 1
Date: Mon, 1 Sep 2008 09:26:51 +1000
From: "Tony Henwood" <AnthonyH <@t> chw.edu.au>
Subject: RE: [Histonet] (para)formaldehyde
To: "MKing" <making <@t> ufl.edu>, <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <B9EAF61856077F47BF9BE2F89AFC555202FB0017 <@t> hedwig.nch.kids>
Content-Type: text/plain; charset="us-ascii"

I agree

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of MKing
Sent: Saturday, 30 August 2008 12:15 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] (para)formaldehyde


The statement that 4% paraformaldehyde solution contains nothing but
water and formaldehyde is simply not true: as has been discussed
thoroughly and often on Histonet (see archives), formaldehyde in aqueous

solution spontaneously decomposes.  This is precisely why formalin
solutions are used, with methanol 'parking' the chemical reaction.
Without measurement one never knows exactly how much formaldehyde is
really present in solutions made from paraformaldehyde, although freshly

made solutions will have concentrations closest to 4%.  It is highly
improbable that 3.8 and 4.0% solutions would produce any detectable and
reproducible differences on any histological procedure, although the
methanol added to formalin might.

Mike King
UF Pharmacology & Therapeutics

-----------------
Message: 5
Date: Wed, 27 Aug 2008 17:26:19 -0400
From: "Monfils, Paul" <PMonfils <@t> Lifespan.org>
Subject: RE: [Histonet] (para)formaldehyde
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<4EBFF65383B74D49995298C4976D1D5E03835C54 <@t> LSRIEXCH1.lsmaster.lifespan.or
g>
Content-Type: text/plain;       charset="iso-8859-1"
10% formalin and 4% paraformaldehyde are interchangeable for most
purposes (in histology at least).  However there are a couple of minor
differences.  First, commercial formaldehyde solution contains 37% to
38% formaldehyde.  Therefore diluting it 1:9 results in a solution
containing 3.7% to 3.8% formaldehyde, while a 4% solution of
paraformaldehyde in water contains a full 4% formaldehyde.  Secondly,
commercial formaldehyde solution contains 10% to 15% methanol as a
preservative.  Therefore diluting it 1:9 results in a solution
containing 1.0% to 1.5% methanol.  This is not a problem for most
histological applications, but it could be a problem in a procedure
where sources of methylation have to be avoided.  4% paraformaldehyde
solution contains no methanol - nothing but water and formaldehyde.

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

Message: 2
Date: Mon, 1 Sep 2008 09:32:17 +1000
From: "Tony Henwood" <AnthonyH <@t> chw.edu.au>
Subject: RE: [Histonet] (para)formaldehyde
To: <rjbuesa <@t> yahoo.com>, <histonet <@t> lists.utsouthwestern.edu>, "MKing"
        <making <@t> ufl.edu>
Message-ID: <B9EAF61856077F47BF9BE2F89AFC555202FB0018 <@t> hedwig.nch.kids>
Content-Type: text/plain; charset="iso-8859-1"

AND
I agree.

Its now getting more precise in the Science.

I would suggest that the NSH put together a committee to determine policy on the nomenclature of formalin solutions so that we can call the lemon the lemon and not the "Sprite" or "Solo" that we seem to have found ourselves in.

Hopefully we can then agree on what we are going to call the stuff.
There is definitely enough publications out there to start from.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Saturday, 30 August 2008 12:28 AM
To: histonet <@t> lists.utsouthwestern.edu; MKing
Subject: Re: [Histonet] (para)formaldehyde


Just for detail sake, methanal (formaldehyde) does NOT decompose in water, it just reacts with it to become methanediol (methylene glycol) and the amount not reacting and remaining as formaldehyde has been estimated in about 0.1% of the total in a 4% formalin solution. The alcohol methanol is added NOT to prevent the formaldehyde hydration, but to as a stabilizer, to retard its polymerization, so it is very likely that between the moment a 4% formaldehyde solution is prepared using paraformaldehyde to the moment it starts to penetrate, bind and cross-link it will be very close to 4% Ren? J.

--- On Fri, 8/29/08, MKing <making <@t> ufl.edu> wrote:

From: MKing <making <@t> ufl.edu>
Subject: [Histonet] (para)formaldehyde
To: histonet <@t> lists.utsouthwestern.edu
Date: Friday, August 29, 2008, 10:14 AM

The statement that 4% paraformaldehyde solution contains nothing but
water and formaldehyde is simply not true: as has been discussed
thoroughly and often on Histonet (see archives), formaldehyde in aqueous
solution spontaneously decomposes.  This is precisely why formalin
solutions are used, with methanol 'parking' the chemical reaction.
Without measurement one never knows exactly how much formaldehyde is
really present in solutions made from paraformaldehyde, although freshly
made solutions will have concentrations closest to 4%.  It is highly
improbable that 3.8 and 4.0% solutions would produce any detectable and
reproducible differences on any histological procedure, although the
methanol added to formalin might.

Mike King
UF Pharmacology & Therapeutics

-----------------
Message: 5
Date: Wed, 27 Aug 2008 17:26:19 -0400
From: "Monfils, Paul" <PMonfils <@t> Lifespan.org>
Subject: RE: [Histonet] (para)formaldehyde
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<4EBFF65383B74D49995298C4976D1D5E03835C54 <@t> LSRIEXCH1.lsmaster.lifespan.org>
Content-Type: text/plain;       charset="iso-8859-1"
10% formalin and 4% paraformaldehyde are interchangeable for most
purposes (in histology at least).  However there are a couple of minor
differences.  First, commercial formaldehyde solution contains 37% to
38% formaldehyde.  Therefore diluting it 1:9 results in a solution
containing 3.7% to 3.8% formaldehyde, while a 4% solution of
paraformaldehyde in water contains a full 4% formaldehyde.  Secondly,
commercial formaldehyde solution contains 10% to 15% methanol as a
preservative.  Therefore diluting it 1:9 results in a solution
containing 1.0% to 1.5% methanol.  This is not a problem for most
histological applications, but it could be a problem in a procedure
where sources of methylation have to be avoided.  4% paraformaldehyde
solution contains no methanol - nothing but water and formaldehyde.

_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet




_______________________________________________
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This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender.

Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead

This note also confirms that this email message has been
virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses.
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------------------------------

Message: 3
Date: Mon, 1 Sep 2008 09:35:08 +1000
From: "Tony Henwood" <AnthonyH <@t> chw.edu.au>
Subject: RE: [Histonet] DAB: old-fashioned style!
To: "Kim Merriam" <kmerriam2003 <@t> yahoo.com>, "Histonet"
        <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <B9EAF61856077F47BF9BE2F89AFC555202FB0019 <@t> hedwig.nch.kids>
Content-Type: text/plain; charset="iso-8859-7"

Try this

DAB Reagent:
1.      Take 40ml Tris Buffer-Wash  and add 6mg of DAB (3,3, Diaminobenzidine Tetrahydrochloride Grade II (Sigma D5637)), mix.
2.      Add 50?l of hydrogen peroxide, mix.
3.      The solution is ready to use.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Kim Merriam
Sent: Saturday, 30 August 2008 3:07 AM
To: Histonet
Subject: [Histonet] DAB: old-fashioned style!


Hi All,

It's been many moons since I had to make DAB by hand and I have lost my recipe.  Can anyone let me know how to make DAB from powder and H2O2?

Thanks,
Kim
 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA



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Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead

This note also confirms that this email message has been
virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses.
**********************************************************************



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

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