From:"Anatech Ltd." <anat...@net-link.net> To:histo...@pathology.swmed.edu Reply-To: Date:Mon, 21 Jun 1999 12:12:19 -0400 Content-Type:text/plain; charset="us-ascii"
A comprehensive review of zinc formalin up to 1992 appears in the following paper: RW Dapson, 1993. Fixation for the 1990's: a review of needs and accomplishments. Biotechnic & Histochem 68:75-82. The mechanism appears to be that zinc ions hold macromolecules in their native conformation via coordinate bonds, preventing the damaging crosslinkages that formaldehyde alone would create. The result is greatly enhanced immunopreservation: rarely is antigen retrieval necessary, and primary antibodies can be diluted 2-10 fold greater than usual. The original use of zinc formalin (by P. Banks and coworkers) was to prevent nuclear bubbling artifact in tissues fixed for less than 24-48 hours. Because nuclear morphology is so sharply defined, zinc formalin is frequently used as a replacement for B-5. Today there are several distinct varieties of zinc formalin, but all share the advantages mentioned above. The original formlula for zinc formalin is 1% zinc sulfate heptahydrate in 10% unbuffered formalin. It tends to precipitate in processors, as the zinc is not soluble in the 70% alcohol used in the first dehydration station. This can cause lines to plug if routine acidic rinses are not performed. Neutral or alkaline water will not dissolve the precipitate. Some processor manufacturers do not want zinc formalin on their machines because of this. The zinc inside the specimens also precipitates (you cannot see it), and causes difficulties in microtomy. Anatech Ltd. developed an unbuffered zinc sulfate formula that does not precipitate in 70% alcohol (it will precipitate if you go directly from it to 80% or higher alcohol), and can be used in all processors. Tissues are not crunchy, and in fact customers often remark that they are easier to cut than those fixed in NBF. All unbuffered zinc formalins are acidic (pH varies from about 3 up to about 4.5). Formalin pigment will develop below about pH 5.3, faster as pH gets lower. The vast majority of our customers using unbuffered zinc formalin do not report formalin pigment artifact (which frankly surprised us), and I can only assume that it is because exposure time is too short for it to happen. Given comparable pH levels, acidic zinc formalin and acidic (unbuffered) formalin will behave similarly regarding pigment formation. Buffered zinc formalin is also available, but again, some formulations precipitate badly in processors. Anatech's product (Z-Fix) is freely soluble in alcohol, and can be made as an alcoholic buffered zinc formalin for those of you who like the added benefits (speed, penetration of fat) that alcoholic formalin provides. Most zinc formalin solutions do not corrode metal any faster than formaldehyde will. Remember that formaldehyde is rather corrosive to nearly all steel, including most forms of stainless (316 and 410 stainless are safe). I suspect that Pearl Gervais' cassette lids are made of some other stainless steel; 316 and 410 are expensive and notoriously difficult to mill. One group of zinc formalin solutions is highly corrosive, however, and that is made with zinc chloride. Faster than its cousins and nearly as aggressive as mercuric chloride, it has been used (not by us) as a B-5 replacement. It can overfix tissues, just like B-5 does, and absolutely must not be put in an enclosed processor. There are very effective rapid, zinc-based B-5 replacements that do not contain the chloride salt. Anatech's products are availble only in the US and Canada. Shandon Lipshaw has a processor-compatible zinc formalin (unbufferd) that is available worldwide. I believe that Richard-Allan's zinc formalin product line is available only in the US (Joan, correct me if I err here!). Sorry for the lengthy answer: I wanted to cover everyone's questions and concerns. Dick Richard W. Dapson, Ph.D. ANATECH LTD. 1020 Harts Lake Road Battle Creek, MI 49015 800-262-8324 or 616-964-6450 Fax 616-964-8084 E-mail anat...@net-link.net http://www.net-link.net/anatech 2010-02-23 TF 发件人: Phebe Verbrugghe 发送时间: 2010-02-23 11:50:40 收件人: Adam . 抄送: histonet 主题: RE: [Histonet] Double labeling with antibodies that need differentfixatives Hello Adam, Thank you very much for this very useful information! Do you know whether this would also work on tissue fixed with formalin instead of zinc buffered formalin by any chance? Also, could you give me the recipe for the zinc formalin and can I use a standard tissue processor for embedding in paraffin or should I use a specific protocol manually and if so, which? Thanks! Phebe ________________________________ From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 10:52 AM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives Although I haven't tried it myself, others have gotten CD31 from BD to work on FFPE tissue using the tyramide amplification system on zinc buffered formalin fixed sections. I've generally had good luck with zinc buffered formalin myself for many antigens so it may work for your other one. See http://www.ncbi.nlm.nih.gov/pubmed/19052548 Just to be clear, they used zinc buffered formalin, which isn't the same thing as zinc fixative. Adam On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe <pverbrug...@meddent.uwa.edu.au> wrote: Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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