4% Paraformaldehyde (PFA) solution preparation

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Paraformaldehyde (PFA) in PBS is one of the widely used fixatives for Immuno-histochemistry (IHC) and fluorescent protein labelled samples. Paraformaldehyde is a polymer of formaldehyde with a wide range of monomeric units typically 8-100. PFA does not have the capacity to fix samples, hence it must be depolymerised in the solution. Heating the PFA powder in the solution leads to its depolymerisation. Although 4% PFA is widely used, there are circumstances where it is used as low as 0.5% to as high as 16%. When dissolved, paraformaldehyde breaks into formaldehyde in solution. Formaldehyde fixes (halts) metabolism by cross-linking protein molecules especially with lysine. It is important to note, that formaldehyde-based fixation is too slow and may take from a few hours to days to fix samples.

PFA is recommended to be made in 1X PBS buffer (neutral buffered formalin; NBA). Neutral pH prevents the formation of formic acid, which is known to form “formalin pigments” in tissue and slower fixation rates.

Below, we have provided a step by step process to prepare 4% PFA solution.

Difference between Formalin, Formaldehyde and Paraformaldehyde

Formaldehyde is a simple aldehyde (equivalent of a monomer to paraformaldehyde) with formula CH2O.

Formalin, on the other hand, is a saturated solution of formaldehyde (37%). 10% formalin is equivalent to 4% formaldehyde. However, many vendors use a small amount of methanol or other chemicals to prevent polymerization of formaldehyde in the solution. These additional reagents must be considered as they may yield unwanted effects.

Paraformaldehyde (PFA) is a polymer of formaldehyde with 8-100 units.

Paraformaldehyde, when dissolved in water, breaks down into formaldehyde. This solution differs from commercially available form (formalin) being relatively pure (devoid of methanol). In immunohistochemistry (IHC) and cell biology experiments, researchers prefer working with PFA solution rather formalin due to the same reason.

Although formalin and paraformaldehyde solutions said to be having formaldehyde, formaldehyde in these solutions is hydrated and converts (most of the formaldehyde) into methylene glycol. In these solutions (formalin or paraformaldehyde), a major portion of methylene glycol is in equilibrium with formaldehyde. However, only formaldehyde (not methylene glycol) have cross-linking ability.

illustration showing formula of formaldehyde and paraformaldehyde

Figure 1. Difference between Formaldehyde and Paraformaldehyde.


Fixation solution for Immuno-histochemistry and fluorescent protein labelled samples.


*Chloramphenicol is optional and is used in order to prevent bacterial contamination.

Table 3. Composition of DRBC agar
ComponentFor 1L media (in grams)
Dextrose (Glucose) 10g
Potassium dihydrogen phosphate 1g
Magnesium sulphate 0.5g or 500mg
Rose Bengal 0.025g or 25mg
Chloramphenicol 0.100g or 100mg
Dichloran 0.002g or 2mg
Agar 15g

Table 3.  Composition of DRBC agar.


Adjust pH 6.9 to 7.4 depending on application with 1N HCl and 1N NaOH.


Here we are describing steps for 4% PFA of the 1L solution. The same trend follows for other volumes as well.

  1. Take 800 mL of 1X PBS.
  2. Add 40 g of Paraformaldehyde powder to 1X PBS.
  3. Stir the mixture at 60˚C in ventilation hood (DO NOT Boil).
  4. PFA powder does not dissolve instantly, you need to raise the pH of the mixture by adding 5N NaOH drop by drop until a clear solution is formed.
  5. There may be small undissolved particles. Cool the solution to room temperature and filter to remove particles.
  6. Adjust the volume to 1L with 1X PBS.
  7. Add 0.5% Triton X-100 (percentage of Triton X-100 varies between 0.01 to 0.5).
  8. Check the pH and adjust with HCl and NaOH if required.
  9. Aliquot into small volumes.


Store PFA solution at room temperature for 1-2 weeks or at 4oC for a few weeks. For long-term storage (up to a year) at -20oC.

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