[Histonet] Decalcification with formic acid sodium citrate
abtdhu at gmail.com
abtdhu at gmail.com
Sun Jul 26 13:33:47 CDT 2015
There was a paper http://www.genedetect.com/Merchant2/ExampleRefs/Decalcifying_protocols.pdf
Talking about formic acid (Morse solution) can get as good result as EDTA in ISH.
FYI.
Dorothy Hu
> Today's Topics:
>
> 1. Re. Decalcification with formic acid sodium citrate
> (Gayle Callis)
> 2. Re: Re. Decalcification with formic acid
>
> Merissa and Tim,
>
>
>
> This formic acid decalcifying solution is basically the classic Evans and
> Krajian fluid (Sheehan and Hrapchak, Theory and Practice of
> Histotechnology, 2nd edition, P.92). Shandon has added other ingredients
> for some reason, and has kept those concentrations proprietary. You really
> don't need to add a surfactant or PVP emulsifier when making up this
> decalcifying agent. Simply use the classic recipe for successful
> decalcification. This is also referred to as buffered formic acid and in
> some publications an "acidic buffer". It is excellent if IHC is needed and
> less damaging, obviously, than a strong mineral HCL acid decalcifiers.
>
>
>
> Sodium citrate crystals (a buffering salt) 10 g
>
> 90% formic acid stock 25 ml
>
> Distilled water 75 ml
>
>
>
> One can calculate the concentration of formic acid i.e. approx. 4.5% since
> is it made from 90% formic acid stock.
>
>
>
> Don't bother with the surfactants or PVP.
>
>
>
> Enjoy an excellent in house formic acid decalcifying solution. I also
> suggest you read Sheehan and Hrapchak textbook chapter on bone as a way to
> familiarize yourself with decalcifiying solutions that manufacturers now
> supply with some modifications. Some manufacturers will refer to these
> methods but probably prefer not to do this since they want you to buy their
> commercial product that is obviously a time saver with elimination of having
> to store stock acid solutions. The classic methods made in house are
> excellent if you have time to make them up. Formic acid with sodium
> formate is another popular buffered formic acid. I suggest you look for
> another source/manufacturer of the your favorite decalcifier in question as
> more than one company will make it. Decal Corp, recently sold to Stat Lab,
> could also be the source as Shandon isn't the only game in town. Others
> are Newcomer Supply, Poly Scientific. Not having to make it up may remain
> your preference.
>
>
>
> Gayle M. Callis
>
> HTL/HT/MT(ASCP)
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> Written by Tim and Merissa:
>
>
>
> Merissa,
>
>
>
> Water 77-80 solvent
>
> Formic acid 21-23 active ingredient
>
> Fluorad >1 surfactant - a
> wetting agent to make the solution wet the bone more easily
>
> Sodium citrate >1 emulsifier , buffer
>
> Polyvinyl pyrrolidone >1 emulsifier
>
>
>
> They say less than one percent of the last three, but you really have no
> idea whether that is 1%, .1% or .01%. It could be any of those.
>
>
>
> But all those surfactants and emulsifiers are meant to keep the solution
> viable for long periods on the shelf. When you make it fresh you don't
> really need them.
>
>
>
> You can either buy a different decalcifier, or make your own. Making your
> own with just the water and acid will work just fine.
>
>
>
>
>
> Tim Morken
>
> Pathology Site Manager, Parnassus
>
> Supervisor, Electron Microscopy/Neuromuscular Special Studies
>
> Department of Pathology
>
> UC San Francisco Medical Center
>
>
>
> -----Original Message-----
>
> From: M.O. via Histonet [mailto:
> <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at
> lists.utsouthwestern.edu]
>
> Sent: Wednesday, July 22, 2015 1:24 PM
>
> To: <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at
> lists.utsouthwestern.edu
>
> Subject: [Histonet] understanding reagents in decalcifier; making it
> in-house
>
>
>
> Hello Histonet
>
>
>
> The supplier for our decalcifier, TBD-2 from Shandon, is having issues with
> getting the product out and we will not be receiving it for at least another
> month. Our samples are piling up and I don't know what I should do, but
> maybe I can make the decalcifier in-house. I am wondering if I can make my
> own based on the reagents they listed and their percentages and if certain
> reagents are not actually necessary.
>
>
>
> The samples we typically decalcify are mouse knees (decal time = 2 days),
> mouse spines (3 days), human bone slabs about 7mm in thickness (7-12 days).
> Fixation is in zinc buffered formalin, then decalcification, then 70% EtOH.
> Our choice to use TBD-2 is due to the gentle decalcification for IHC and we
> get GREAT results.
>
>
>
> Composition of Shandon TBD-2 Decalcifier:
>
> Component Weight %
>
> Water 77-80
>
> Formic acid 21-23
>
> Fluorad >1
>
> Sodium citrate >1
>
> Polyvinyl pyrrolidone >1
>
>
>
> If you have any input on what reagents I should use and the percentages for
> making a decalcifier myself, it would be much appreciated!
>
>
>
> Thank you for you help,
>
> Merissa
>
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Sat, 25 Jul 2015 10:54:59 -0700
> From: Merissa <modz9636 at gmail.com>
> To: "gayle.callis at bresnan.net" <gayle.callis at bresnan.net>
> Cc: Histonet <histonet at lists.utsouthwestern.edu>
> Subject: Re: [Histonet] Re. Decalcification with formic acid sodium
> citrate
> Message-ID: <7E946DAD-9E26-43CB-B822-31D180C8CB71 at gmail.com>
> Content-Type: text/plain; charset=us-ascii
>
> Thank you, Gayle! This is exactly what I was looking for and we are willing to make this in house. We are trying just for if acid and water, but the buffering salt should be added. I will try de calcifying mouse knees next week with this protocol. Thank you for the reference, I appreciate your help!
>
> Sincerely,
> Merissa
>
>
>
>> On Jul 25, 2015, at 10:24 AM, Gayle Callis via Histonet <histonet at lists.utsouthwestern.edu> wrote:
>>
>> Merissa and Tim,
>>
>>
>>
>> This formic acid decalcifying solution is basically the classic Evans and
>> Krajian fluid (Sheehan and Hrapchak, Theory and Practice of
>> Histotechnology, 2nd edition, P.92). Shandon has added other ingredients
>> for some reason, and has kept those concentrations proprietary. You really
>> don't need to add a surfactant or PVP emulsifier when making up this
>> decalcifying agent. Simply use the classic recipe for successful
>> decalcification. This is also referred to as buffered formic acid and in
>> some publications an "acidic buffer". It is excellent if IHC is needed and
>> less damaging, obviously, than a strong mineral HCL acid decalcifiers.
>>
>>
>>
>> Sodium citrate crystals (a buffering salt) 10 g
>>
>> 90% formic acid stock 25 ml
>>
>> Distilled water 75 ml
>>
>>
>>
>> One can calculate the concentration of formic acid i.e. approx. 4.5% since
>> is it made from 90% formic acid stock.
>>
>>
>>
>> Don't bother with the surfactants or PVP.
>>
>>
>>
>> Enjoy an excellent in house formic acid decalcifying solution. I also
>> suggest you read Sheehan and Hrapchak textbook chapter on bone as a way to
>> familiarize yourself with decalcifiying solutions that manufacturers now
>> supply with some modifications. Some manufacturers will refer to these
>> methods but probably prefer not to do this since they want you to buy their
>> commercial product that is obviously a time saver with elimination of having
>> to store stock acid solutions. The classic methods made in house are
>> excellent if you have time to make them up. Formic acid with sodium
>> formate is another popular buffered formic acid. I suggest you look for
>> another source/manufacturer of the your favorite decalcifier in question as
>> more than one company will make it. Decal Corp, recently sold to Stat Lab,
>> could also be the source as Shandon isn't the only game in town. Others
>> are Newcomer Supply, Poly Scientific. Not having to make it up may remain
>> your preference.
>>
>>
>>
>> Gayle M. Callis
>>
>> HTL/HT/MT(ASCP)
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Written by Tim and Merissa:
>>
>>
>>
>> Merissa,
>>
>>
>>
>> Water 77-80 solvent
>>
>> Formic acid 21-23 active ingredient
>>
>> Fluorad >1 surfactant - a
>> wetting agent to make the solution wet the bone more easily
>>
>> Sodium citrate >1 emulsifier , buffer
>>
>> Polyvinyl pyrrolidone >1 emulsifier
>>
>>
>>
>> They say less than one percent of the last three, but you really have no
>> idea whether that is 1%, .1% or .01%. It could be any of those.
>>
>>
>>
>> But all those surfactants and emulsifiers are meant to keep the solution
>> viable for long periods on the shelf. When you make it fresh you don't
>> really need them.
>>
>>
>>
>> You can either buy a different decalcifier, or make your own. Making your
>> own with just the water and acid will work just fine.
>>
>>
>>
>>
>>
>> Tim Morken
>>
>> Pathology Site Manager, Parnassus
>>
>> Supervisor, Electron Microscopy/Neuromuscular Special Studies
>>
>> Department of Pathology
>>
>> UC San Francisco Medical Center
>>
>>
>>
>> -----Original Message-----
>>
>> From: M.O. via Histonet [mailto:
>> <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at
>> lists.utsouthwestern.edu]
>>
>> Sent: Wednesday, July 22, 2015 1:24 PM
>>
>> To: <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at
>> lists.utsouthwestern.edu
>>
>> Subject: [Histonet] understanding reagents in decalcifier; making it
>> in-house
>>
>>
>>
>> Hello Histonet
>>
>>
>>
>> The supplier for our decalcifier, TBD-2 from Shandon, is having issues with
>> getting the product out and we will not be receiving it for at least another
>> month. Our samples are piling up and I don't know what I should do, but
>> maybe I can make the decalcifier in-house. I am wondering if I can make my
>> own based on the reagents they listed and their percentages and if certain
>> reagents are not actually necessary.
>>
>>
>>
>> The samples we typically decalcify are mouse knees (decal time = 2 days),
>> mouse spines (3 days), human bone slabs about 7mm in thickness (7-12 days).
>> Fixation is in zinc buffered formalin, then decalcification, then 70% EtOH.
>> Our choice to use TBD-2 is due to the gentle decalcification for IHC and we
>> get GREAT results.
>>
>>
>>
>> Composition of Shandon TBD-2 Decalcifier:
>>
>> Component Weight %
>>
>> Water 77-80
>>
>> Formic acid 21-23
>>
>> Fluorad >1
>>
>> Sodium citrate >1
>>
>> Polyvinyl pyrrolidone >1
>>
>>
>>
>> If you have any input on what reagents I should use and the percentages for
>> making a decalcifier myself, it would be much appreciated!
>>
>>
>>
>> Thank you for you help,
>>
>> Merissa
>>
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet at lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
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