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Douglas W. Cromey, M.S.
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Formaldehyde Fixatives © 2002 – 2012 , The University of Arizona Page 1 Formaldehyde Fixatives Douglas W. Cromey, M.S. – Cellular Imaging Facility Core, Southwest Environmental Health Sciences Center (NIEHS grant # ES 0 06694) Staff – Cell Biology & Anatomy Histology Service Lab, University of Arizona, Tucson, Arizona Informati on about 37% Formaldehyde There is no definitive age after which 37% Formaldehyde is no longer useful as a stock solution. Formaldehyde chemistry is moderately complex, but after discussions with other microscopists, m anufacturers and reviewing pertinent texts, the following observations are applicable. Formaldehyde should be stored at room temperature (cold temperatures encourage the formation of trioxymethylene with a resulting white precipitate) Formaldehyde should be stored tightly sealed, since expo sure to air encourages the oxidation of formaldehyde to formic acid (37% formaldehyde is usually shipped with 10 – 15% methanol to inhibit this change) . Our recommendation regarding 37% stock solutions: If a solution of 37% formaldehyde is clear, colorles s and has no precipitate, and has been stored at room temperature in a tightly sealed bottle that has not been exposed to sunlight, it should be good. However, we still do not recommend using a stock bottle that is older than 1 year. Bottles of 37% forma ldehyde that are already opened should not be used more than six months. Consequently, we recommend that labs purchase their formaldehyde more frequently and in smaller quantities than perhaps they have done in the past. Use of 37% Formaldehyde is not re commended for electron microscopy fixatives. A better choice is to use a higher grade, methanol – free formaldehyde , or a fresh solution made from paraformaldehyde (see further comments below) . Information about 10% Formalin The fixative 10% buffered form alin is commonly used to preserve tissues for routine histology in many labs. The formaldehyde has a greater chance for oxidation in this concentration of tissue fixative and eventually the solution will start to drop in pH, in spite of the buffer. We rec ommend that 10% buffered formalin solutions be used no longer than 3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5. The other problem with 10% buffered formalin is the slowly increasing concentration of methanol (an unwanted byproduct of aging formaldehyde) . Methanol promotes clumping of proteins, instead of the cross – linking of proteins that formaldehyde performs. A methanol – free fixative will give the best prese rvation, particularly if you plan to use the tissue for antibody staining at a later time. The most common way to avoid methanol in a formaldehyde solution is to make the solution up fresh from crystalline paraformaldehyde. Paraformaldehyde can be quite hazardous to handle and it is often difficult to get it to go into solution. If your lab is not a regular user of formaldehyde fixatives, there are a couple of easier options that we recommend. One option is to purchase methanol – free formaldehyde (aq) in sealed ampoules. Simply add PBS to achieve the correct formaldehyde concentration and use immediately. Ten 10 ml ampoules of 16% methanol – free formaldehyde costs approximately $2 7 . (See sample protocol at the end of this document) The other option i s to buy 10% neutral buffered Formalin (4% formaldehyde) from a scientific supply house, use it for 3 – 6 months and then discard it (as a hazardous material) . There will be some methanol in this solution (typically 1 – 2%) , but if used soon after purchase thi s should not be significant for most users. The buffered solution helps slow the acidification process. A one liter bottle costs approximately $20 – 25. Store the fixative at room temperature. Local readers of this page should contact the CBA Histology Se rvice Lab (626 – 4415) if you have trouble locating a supplier. (Please note different suppliers use different buffering solutions. For consistent immunohistochemistry or immunofluorescence , users should stick with one supplier.)
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