Zbigniew Darzynkiewicz

fixation

Paraformaldehyde is a polymerized form formaldehyde. It is hardly soluble and it cannot be used as a fixative. Only formaldehyde is used as a fixative. Howeve, formaldehyde in aqueous solutions spontaneously polymerizes. Therefore, methanol is often added to slowdown the polymerization reaction. Solutions of formaldehyde (usually ~ 37%) in water, containig 10-15 % methanol as a preservative are generally called "formaldehyde"; such solutions are being sold by most reagent companies. Solutions further diluted (4-10 %) received name "formalin". Methanol-free formaldehyde, which sometimes is preferred (e.g. for fixing cells for some some histochemical reactions or in immunocytochemistry), can be obtained by hydrolysis of paraformaldehyde. This is usually done by extensive heating of paraformaldehyde solutions. Because of this procedure the methanol-free formaldehyde received (incorrrectly) the name "paraformaldehyde". In the past, this was the most common way to obtain methanol-free formaldehyde. Unfortunately, this incorrect name is still often used in the literature, generating the confusion. The methanol-free formaldehyde solutions can now be purchased. Some are called "ultrapure". We purchase such solutions (10%) from Polysciences, Inc. (800-523- 2575); they can be stored at room temperature. I would not recommend, however, to store them longer than one year, since formaldehyde in these solutions still has tendency to polymerize. It should be noted that all formaldehyde solutions are highly toxic and carcinogenic.

Catherine, Autofluorescence is usually higher with older aldehyde solutions. I would recommend using freshly prepared formaldehyde (less than one month old). Also, the addition of 0.3% Triton X- 100 to the antibody incubation solutions will decrease autofluorescence. --Randy

Hi Catherine: Formaldehydes and glutaraldehydes are bad actors when it comes to fluorescence-based cytologic analysis. Either get away from fixation with aldehydes or the answer to the problem is aldehyde blocking. This is done by reducing the -CHO groups to -OH with sodium borohydride or by usingbland amino groups (glycine, bovine albumin, skimmed milk). The other option is to freeze cells. Make cytospins when you need them. The third option is to live with the autofluorescence. Just dedicate FITC channel to autofluorescence. Autofluorescence Eliminator Reagent http://www.chemicon.com/featured/autofluorescence.asp http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.p df Good luck Padma ------Hi Catherine, One method is to use the violet laser for autofluorescence correction. The blue laser excites both autofluorescence and FITC/Alexa 488. The violet laser excites only the autofluorescence. Subtracting the violet excited green scan images from the FITC/Alexa 488 scan images leaves a close approximation to the specific fluorescence. Best wishes, Ed ------Dear Catherine, This is a common problem in immuno-histology. Attached is a paper that may help. I have not tried sodium borohydride in a flow setting yet, but one of the researchers who use my flow lab is going to give it a go. Regards Rob W

Paraformaldehyde is a white solid polymer (HCHO)n of formaldehyde where n is at least 6. It is used as a disinfectant, fumigant, and fungicide. It is formaldehyde (HCHO), that is used as cells/tissues fixative, not "paraformaldehyde". Because formaldehyde in solution undergoes oxidation and polymerization, it is common to make fresh solutions by depolymerizing paraformaldehyde, most frequently by heating in water solution. Hence, such product is often (incorrectly) called "paraformaldehyde". The hydrolysis is a hazardous procedure, because paraformaldehyde/formaldehyde vapours are strongly irritating and carcinogenic. To make solutions of formaldehyde more stable methanol is often added, and this is called "formalin". Formalin 100% is a soluition of 37% by wt of formaldehyde gas in water, with 10-15% methanol to prevent polymerization. It is also often called "formalin 40", as it contains about 40g of formadehyde in 100 g of water. For most applications that call for "paraformaldehyde" we use the methanol-free formaldehyde, that is available from Polysciences, Inc (Warrington, PA). I do not know how they stabilize their product, but we store it up to a year, as prescribed by the vendor, and do not see any deterioration of the results.

Zbigniew Darzynkiewicz, M.D., Ph.D. Brander Cancer Research Institute