[Histonet] IHC on paraformaldehyde-fixed

[Nicole Collette]

Hi, All,

I have recently started doing IHC on whole-mount embryos, and one way 
to fix them at harvest is in a typical formaldehyde/formalin 
fixative, followed by short- or long-term storage in 100% methanol. 
The alternative fixation protocol, for antigens that are sensitive to 
formaldehyde/formalin, is to fix them in 4:1 methanol: DMSO- followed 
again by short- or long-term storage in methanol. (BTW the protocol 
works, at least for the antigens I have tested in my limited 
experience). I guess a counterpoint to other comments...

CSH protocols; 2008; doi: 10.1101/pdb.prot 4820

Nicole Collette
LLNL
postdoc


>Good point. I never tested it directly side-by-side to check for the 
>effects of 1% methanol directly. It could be old-fashioned paranoia. 
>However, it was a variable I wanted to omit as I know methanol (we 
>are talking 100%) has deleterious effects to some mouse antigens. 
>Therefore, it wasn't something I wanted to risk ... particularly 
>since 95% of what I do now is bone/hematopoietic tissue work anyway 
>which can be a nightmare to begin with.
>
>
>
>>Interesting point.
>>Since 10% buffered formalin (made from the concentrated 38%
>>formaldehyde) contain about 1% methanol, has it been shown that this has
>>a deleterious effect on ANY antigens or are we expecting this worse case
>>senario as being the norm?
>>
>>I am not aware of any antigens (or antigen-antibody combination) that
>>has been badly effected by 10% formalin that is NOT effected by 10%
>>formaldehyde. Are you aware of any??
>>
>>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, AUSTRALIA
>>
>>
>>
>>
>>-----Original Message-----
>>From: anh2006 <@t> med.cornell.edu [mailto:anh2006 <@t> med.cornell.edu]
>>Sent: Friday, 5 December 2008 1:31 AM
>>To: Tony Henwood; Jan Shivers; histonet
>>Subject: Re: [Histonet] IHC on paraformaldehyde-fixed
>>
>>
>>So true. However, be aware that 10% neutral buffered formalin we use has
>>methanol in it which may affect certain antigens so there may be some
>>difference in staining (hence why for mouse work we now only use 4% PFA
>>in pure PBS). It is good to be aware of the other ingredients in your
>>fixative solutions, whether commercially prepared or a homemaede recipe,
>>as it isn't only the formaldehyde fixative which can make a difference.
>>
>>
>>-----Original Message-----
>>From: Tony Henwood <AnthonyH <@t> chw.edu.au>
>>
>>Date: Thu, 04 Dec 2008 09:35:09
>>To: Jan Shivers<shive003 <@t> umn.edu>;
>>histonet<histonet <@t> lists.utsouthwestern.edu>
>>Subject: RE: [Histonet] IHC on paraformaldehyde-fixed
>>
>>
>>Gee I hate the term paraformaldehyde (as many of you probably know)
>>
>>This is an example of how confusion of terms can cause unnecessary work.
>>Is "4% paraformaldehyde" different from 4 % formaldehyde?
>>
>>No
>>
>>Should any procedure done to tissues fixed in "4% paraformaldehyde" give
>>results different to those fixed in 4% formaldehyde or 10% formalin?
>>
>>No since they are the same thing.
>>
>>
>>As Manoonkitiwongsa and Schultz (Histochem J 34: 365-367, 2002) state
>>when paraformaldehyde actually becomes a fixative, it is no longer
>>paraformaldehyde by chemistry or fixation capacity. Rather, it is
>>formaldehyde in water without methanol or any other stabiliser. Without
>>heat and an alkaline environment, paraformaldehyde in water is simply a
>>paraformaldehyde suspension with little fixation capacity. If the
>>fixative is prepared from paraformaldehyde then it should be termed 4%
>>formaldehyde freshly prepared from paraformaldehyde. If a concentrated
>>formalin solution (40% formaldehyde) is used, then it should be termed
>>10% formalin.
>>
>>If you do a search on Histonet for paraformaldehye, you will find that
>>this topic has been extensively discussed.
>>
>>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, AUSTRALIA
>>
>>
>>
>>
>>-----Original Message-----
>>From: histonet-bounces <@t> lists.utsouthwestern.edu
>>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jan
>>Shivers
>>Sent: Thursday, 4 December 2008 8:34 AM
>>To: histonet
>>Subject: [Histonet] IHC on paraformaldehyde-fixed
>>
>>
>>Has anyone ever done IHC on parafomaldehyde-fixed tissues, and if so,
>>how well did it work?  Will the same antigen-retrieval methods used with
>>formalin-fixed tissue be applicable?
>>
>>I'm asking for an investigator, who already has his tissues fixed in
>>paraformaldehyde.
>>
>>Jan Shivers
>>Senior Scientist
>>Pathology Teaching Program
>>Histology/IHC/EM Section Head
>>University of Minnesota
>>Veterinary Diagnostic Laboratory
>>1333 Gortner Ave.
>>St. Paul, MN  55108
>>612-624-7297
>shive003 <@t> umn.edu
>--
>
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