Polymethanal (paraformaldehyde)

Polymethanal is a common fixative in biology. It is used to reduce degradation in cells, tissue and entire organisms before further experiments, e.g. antibody staining.

Recipes for polymethanal buffers

Some people make 16 or 24% stocks to be diluted at a later point. But the solid polymethanal / PFA is very hard to dissolve at these high concentrations. Stocks or working solutions can be preserved by freezing for later use. Otherwise, methanal reacts with itself to form methanol and methanoate (acidification).

PFA 4%

for 50ml

2.0 g PFA
+5 ml H2O
+drop of NaOH (0.5-1.0M)
+45 ml PBS
(correct pH to 7.4 with HCl)

PFA 1%

for 10ml

0.1 g paraformaldehyde powder in small glass tube
+0.5 ml distilled water
+drop of 0.5-1.0M sodium hydroxide

heat to ~80°C for 2-3 mins
shake in water bath until PFA dissolved (beaker of very hot water)

+9.5ml PBS
correct pH with HCl if necessary

Can also be solved by heating to 80°C for 3 hours.

Too many names

There's plenty of confusion regarding this chemical because too many synonyms exist. Paraformaldyhde is probably still the most common term among biologist, but it ignores attempts to introduce meaningful and systematic names in chemistry. Polymethanal is clearer: poly - polymer, meth - single carbon, -al - aldehyde group.

Here's a list of equivalent terms: polymethanal, polyoxymethylene, polyformaldehyde, paraformaldehyde, paraform, polytrioxane,.. (regards from Babel) Common abbreviations are PFA, pMeO.

CAS number = 30525-89-4

Comparison with methanal

Polymethanal is often preferred over methanal which is also used in fixation of live material. This is because polymethanal is a solid which makes it easier to transport and a little safer to use. The chemistry of fixation is similar due to the partial depolymerisation of polymethanal in the buffer making process (basic hydrolysis).

Chemistry of fixation

The aldehyde group, especially of methanal, is very reactive. It readily combines with amino groups to amides or with alcohol groups to esters. These reactions are mostly irreversible. Unreacted molecules have to be removed prior to subsequent experiments since they easily damage proteins like antibodies.

Problems with Fixation

There are issues in fixing free GFP in the cytoplasm of cells. Firstable 4% PFA reduces GFP fluorescence to some extend, but it is tolerable for most experiments. 4% PFA can also destroy the subcellular localization of GFP, i.e. exclusion from the nucleus. In our hands the fixation with more than 2% PFA or in combination with 0.5%< glutaraldehyde or 0.5%< acrolein destroys inclusion/exclusion of IRF3-GFP from the nucleus. Moreover 2% PFA fixed cells expressing IRF3-GFP loose integrity after 2-5 hours post fixation during storage in PBS 1x, meaning that cytoplasm containing GFP leaks out of the cells and remains as a bubble outside the cell. It can be washed away but does not distribute in the PBS buffer on its own. This issue was not solved by higher/lower PFA concentrations during fixation, by storage in H2O/PBS1x/PBS2x/fixation solution/permeabilization of cells after fixation with Tx100 or Saponin 0,2%.

Polymethanal safety data sheets

• EMD Chemical/Merck
• Oxford Uni Chemistry

Links

Wikipedia

• methanal = formaldehyde
• polymethanal