Dear Juan, Paraformaldehyde & formaldehyde are the same chemical. You might like to think of them as the solid & gas phases respectively. Formaldehyde solution does contain methanol as a stabilising agent, a preservative. This means that formaldehyde can be prepared & stored in bulk. Over time the formaldehyde molecules polymerise, this gives rise to the white precipitate that can be seen in the bottom of formalin solutions. The methanol in the solution can damage/cover some antigenic sites Paraformaldehyde is my choice of fixative for immunological work, usually a 2 -4 % solution in buffer. Preferably a buffer that is adjusted for both pH & osmolarity. I prefer PIPES or HEPES buffer for specialist histological or electron microscopy studies, but PBS is far more cost effective especially for flow work. The PBS I run in my flow is not checked for osmolarity. The problem with paraformaldehyde solutions is that they begin to polymerise as soon as you make them up. Always use paraformaldehyde as a fresh solution. I don't use it if it is more than 24 hours old, but some find it is still OK after a week. Methanol has a different fixative action to aldehydes, it is a precipitates proteins. Generally speaking it is an agent that causes shrinking. Therefore, yes it may create altered scatter properties, & yes it may limit some antigenic selection. It should be noted that aldehyde fixatives can also cause modification to cell size & shape through the action (or inaction) of your chosen buffer, & some antigens are vey sensitive to aldehyde fixation. I have never considered the action of formic acid. But, I would never use a fixative without a good buffer. I assume the action of the formic acid would damage some antigenic sites & cellular constituents, so changes to scatter properties may be seen. Hope this helps. Regards Rob W. At 08:37 PM 2/23/99 -0800, you wrote: > >HI > >Are differences in the flourescence, scater propieties or antigenic >detection if one use formaldehyde for fixed cells ? > >I know that formaldehyde is a cross-linking fixative, forming reversible >methylene bridges between amino, imino, sulfhydryl, and hydroxyl groups >within proteins.Also reacts to a lesser extent with lipids and >carbohydrates, but, inlike alcoholic fixatives, does not act as a >permeabilizing agent. (Clevenger C, Shankey V.: Cytochemistry II: >Immunofluorescence measurement of intracellular antigens. In Clinical >Flow Cytometry, principles and aplication. Bauer et al ED pp 158.) > >Also the oxydation of formaldehyde results in the presence of formic >acid , and methanol is found as a stabilizing agent in unbuffered >solutions of formaldehyde. > >Does the methanol afect flourescence, scater propieties or antigenic >detection ? > >Does de formic acid afect flourescence, scater propieties or antigenic >detection ? > >Does th formaldehyde afect the satining of intracellular antigens ? > >I hope yours ideas. > > > >JUAN LUIS CASTILLO N. >LABORATORIO DE CITOMETRIA DE FLUJO >HOSPITAL DEL TRABAJADOR DE CONCEPCION >CARDENIO AVELLO 36 >CONCEPCION >CHILE >PHONE: 56-41-201722 >FAX: 56-41-201708 >EMAIL: axelyoyi@entelchile.net > > R. Wadley, B.App.Sc, M.L.S Laboratory Manager Cellular Analysis Facility School of Microbiology & Immunology UNSW, New South Wales, Australia, 2052 Ph (BH) +61 (2) 9385 3517 Ph (AH) +61 (2) 9555 1239 Fax +61 (2) 9385 1591 E-mail r.wadley@unsw.edu.au www http://www.unsw.edu.au/clients/microbiology/CAF.html (Under development)
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