4% Paraformaldehyde (PFA) solution preparation

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    Introduction

    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

     Applications

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

    pH

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

    Composition

    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

    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 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.

    Storage

    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.

    References

    1. Cold Spring Harbor Protocols.
    2. BOSTER.

    2 Comments

    1. Ephrem Zegeye on April 12, 2021 at 6:18 pm

      Hi,
      Thank you for this excellent protocol. I wonder about the purpose of Triton at step 7,or is it typo?
      Best regards,
      Ephrem

      • Sharebiology on April 13, 2021 at 11:18 am

        Hello Ephrem Zegeye,

        Thank you for this excellent protocol.

        Glad you liked the post.

        I wonder about the purpose of Triton at step 7,or is it typo?

        It is not a typo. The fixative diffuses fast (due to small size) into tissue. However, the rate of diffusion is further enhanced by using detergents like Triton. Triton make pores in membranes. These pore allow diffusion of PFA (actually methylene glycol – soluble form of formaldehyde) much faster than fixative without detergent. Most of the Researchers use 0.05% triton X100. But the percentage varies depending on sample type.

        Hope this help

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