OVERVIEW

A stock solution of 16% paraformaldehyde can be prepared by dissolving 16 g of paraformaldehyde in water to a final volume of 100 ml. This stock solution can be used to prepare fixative solutions for immunocytochemistry and immunohistochemistry. A 16% paraformaldehyde solution can also be purchased from several suppliers.

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REQUIREMENTS

Reagents and solutions
♦ Paraformaldehyde powder
♦ 1M Sodium hydroxide (NaOH)
♦ 1M Hydrogen Chloride (HCl) (optional)
♦ Deionized/double distilled water/MiliQ water *

* Depending on the downstream application, you can choose the water quality.

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

COMPOSITION:
16% paraformaldehyde in water

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

PROCEDURE: Preparation of 16% Paraformaldehyde Solution in Water

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 16 g of paraformaldehyde in 80 ml deionized/Mili Q water.
♦ Weigh out 16 g paraformaldehyde in a 250 ml conical flask or beaker.
♦ Add 80 ml of deionized/Mili Q water. Cover the conical flask with parafilm or aluminum foil.
♦ Add a magnetic bead and place the beaker on a magnetic stirrer. Set the temperature of the magnetic stirrer to 60°C. Add a few drops of 1M NaOH solution (0.1 ml), and mix it with moderate stirring until the solution becomes clear. 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.

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 deionized/Mili Q water.
♦ Transfer the solution to a measuring beaker and add deionized/Mili Q water 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 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/2010/1/pdb.rec12102.full
• 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