OVERVIEW

• A 4% paraformaldehyde in PBS is frequently used to fix biological samples.
• Paraformaldehyde is a white crystalline solid powder, which is nothing but a polymeric form of formaldehyde. 
• Formaldehyde-based fixatives (e.g., paraformaldehyde, Formalin) are best known for their ability to preserve the morphology of biological samples. However, their fixation mechanism mainly relies on cross-linking which can destroy the antigenicity of epitopes, resulting in the loss of antigen-antibody reaction. 
• Freshly prepared 4% paraformaldehyde solution offers much more pure formaldehyde fixative than formalin. Formalin, which is also used as an alternative to formaldehyde fixative, contains 10 – 15% methanol as a stabilizer.
• A 100 ml of 4% (w/v) paraformaldehyde can be prepared by dissolving 4g of paraformaldehyde in PBS to a final volume of 100 ml.

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REQUIREMENTS

Reagents and solutions
♦ Paraformaldehyde powder
♦ 1M Sodium hydroxide (NaOH)
♦ 1M Hydrogen Chloride (HCl) (optional)
♦ Phosphate-Buffered Saline (PBS), pH 7.6 *

* PBS can be available as 10X concentrated stock solution. Dilute it to 1x PBS before use.

Equipment and disposables
♦ Fume hood
♦ Magnetic stirrer with temperature control
♦ Conical flask / Beaker
♦ Aluminum foil / Parafilm
♦ 15 ml tubes / 50 ml tubes
♦ Spatula

COMPOSITION:
4% paraformaldehyde in 1x PBS

OBJECTIVE:
Preparation of 100 ml of 4% paraformaldehyde solution in PBS

PROCEDURE: Preparation of 4% Paraformaldehyde Solution in PBS

Use appropriate personal protective equipment (lab coat, gloves, goggles, etc) for your safety and follow your institute’s guidelines. You must read the material safety data sheet (MSDS) before working with any chemical.

Step 1: Dissolve 4 g of paraformaldehyde in 80 ml PBS.
♦ Weigh out 4 g paraformaldehyde in a 250 ml conical flask or beaker.
♦ Add 80 ml of PBS. Cover the conical flask with parafilm or aluminum foil.
♦ Place the beaker on a magnetic stirrer. Set the temperature of the magnetic stirrer to 60°C. Mix it with moderate stirring until the solution becomes clear.
♦ If the solution is not clear in 1 h, add a few drops of 1M NaOH. It may take a few hours to dissolve paraformaldehyde completely.

Notes:
1. At the start of mixing, the solution will appear milky.
2. Heating is necessary to dissolve the paraformaldehyde completely in the solution.

Tips:
1. If you don’t have a magnetic stirrer with a heating facility, keep the solution in a 60°C water bath and occasionally swirl the solution. This can take a long time to dissolve paraformaldehyde.
2. A slightly alkaline pH is required to dissolve the paraformaldehyde completely in the solution. Use PBS with pH 7.6.

Precautions:
1. Paraformaldehyde is toxic.
2. Exposure to paraformaldehyde powder or vapours can cause serious damage.

Step 2 (optional): Depending on the requirement, the pH of the solution can be adjusted using 1M HCl.

Step 3: Adjust the volume to 100 ml with PBS.
♦ Transfer the solution to a measuring beaker and add PBS to bring the final volume to 100 ml.
♦ Transfer back to the beaker and mix again for a minute.

Step 4: Filter the solution by passing it through a filter paper (Whatman No. 1 filter paper) to remove undissolved particles.

Storage:
Store the solution at -20°C in small aliquots depending on the use. The solution can also be stored at 4°C for a limited time (2 weeks). 

Note:
Storing the solution at room temperature will lead to the formation of formic acid in the solution.

Tips:
1. Do not thaw the solution repeatedly. Once the solution is thawed, use it completely. Avoid repeated thawing to maintain the quality of fixation of biological specimens.
2. If you see a white precipitate upon thawing, keep the solution on the 60°C water bath until the solution becomes clear.

REFERENCES:

• http://cshprotocols.cshlp.org/content/2006/1/pdb.rec9959.short
• https://www.rndsystems.com/resources/protocols/protocol-making-4-formaldehyde-solution-pbs

FURTHER READINGS

• https://www.sciencedirect.com/topics/medicine-and-dentistry/paraformaldehyde
• http://olsonlab.com/resources/Data-Files/OlsonLabProtocol.PFA_Preparation.pdf