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.
- Fixation solution for Immuno-histochemistry and fluorescent protein labelled samples.
Adjust pH 6.9 to 7.4 depending o application with 1N HCl and 1N NaOH.
|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|
- 1N HCl.
- 1N NaOH.
- 1X PBS (Click here to know how to make).
- Paraformaldehyde powder.
Material and instruments required:
- Glass Beaker.
- Filter unit.
- Hotplate magnetic stirrer and a magnetic bead.
- Ventilation hood.
- Pipettes and Tips.
- pH meter.
Here we are describing steps for 4% PFA of the 1L solution. The same trend follows for other volumes as well.
- Take 800 mL of 1X PBS.
- Add 40 g of Paraformaldehyde powder to 1X PBS.
- Stir the mixture at 60˚C in ventilation hood (DO NOT Boil).
- 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.
- There may be small undissolved particles. Cool the solution to room temperature and filter to remove particles.
- Adjust the volume to 1L with 1X PBS.
- Add 0.5% Triton X-100
- Check the pH and adjust with HCl and NaOH if required.
- 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 -20o C.