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4% paraformaldehyde (PFA) fixative


Heat to 60C a volume of water equal to slightly less than 2/3 the desired final volume of fixative. Weigh out a quantity of paraformaldehyde (Baker) that will make a 4% solution and add it with a stir bar to the water. Cover. Transfer to fume hood and maintain on heating plate at 60C with stirring. Add 1 drop 2 N NaOH with a Pasteur pipet. The solution should become almost clear fairly rapidly, but will still have some fine particles that will not go away. Be careful not to overheat the solution. Remove from heat and add 1/3 vol 3 PBS. Bring pH of solution to 7.2 with HCl, add water to final volume, and filter using a Millipore or Nalgene filter. Cool to room temperature, or to 4C on ice.


CAUTION: Formaldehyde is a carcinogen and may cause allergic reactions.


Formaldehyde in liquid form (formalin; 37%) contains impurities such as methanol (6% to 15%) and formic acid (<0.05%), which make it unsuitable for many histochemical applications. Formaldehyde solution for these purposes is best prepared from paraformaldehyde, which is heated at >65C to break the polymer into formaldehyde. Formaldehyde prepared from paraformaldehyde may either be used soon after preparation or stored as a stock solution for preparation of relatively fresh fixative solutions.


CAUTION: Formaldehyde is toxic, and the preparation process involving heating results in considerable vaporization, which increases the hazard. It is essential to use appropriate safety procedures, such as working in a fume hood.