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4% Paraformaldehyde (PFA) solution preparation

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.

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

Applications

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

pH

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

Composition

4% Paraformaldehyde (PFA) fixative composition and preparation
Keep the pH between 7.2 to 7.4 using HCl and NaOH. 0.5% Triton X-100
4% Paraformaldehyde (PFA) fixative composition and preparation
Keep the pH between 7.2 to 7.4 using HCl and NaOH. 0.5% Triton X-100
Reagent Add for 100 ml Add for 500 ml Add for 1 L
Paraformaldehyde 4 g 20 g 40 g
1X PBS 80 ml + 20ml 400 ml + 100 ml 800 ml + 200 ml

Click here to get molarity and dilution calculator for making stocks

Reagents required:

Material and instruments required:

  • Glass Beaker.
  • Filter unit.
  • Hotplate magnetic stirrer and a magnetic bead.
  • Ventilation hood.
  • Pipettes and Tips.
  • pH meter.
  • Thermometer.

Procedure:

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 1N 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
  8. Check the pH and adjust with HCl and NaOH if required.
  9. Aliquot into small volumes.

Storage

At room temperature, for 1-2 weeks. At 4oC for a few weeks. For long term storage (up to a year) at -20o C.

References

  1. Cold Spring Harbor Protocols.
  2. BOSTER.
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