Preparation of 4% Paraformaldehyde Solution in PBS

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 pure formaldehyde fixative than formalin. Formalin, which is also used as an alternative to formaldehyde fixative, contains 10 – 15% methanol as a stabilizer.

REQUIREMENTS

Reagents and solutions
♦ Paraformaldehyde powder
♦ Phosphate-Buffered Saline (PBS)

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

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

PROCEDURE

Step 1: Dissolve 4 g of paraformaldehyde in 80 ml 1x PBS.
♦ Weigh out 4 g paraformaldehyde in 250 ml conical flask or beaker.
♦ Add 80 ml of 1X PBS. Cover the conical flask with parafilm or aluminum foil.
♦ Place the beaker on a magnetic stirrer. Set the temperature of the magnetic stirrer at 60°C. Mix it with moderate stirring until the solution becomes clear.

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.
2. A slightly alkaline pH is required to dissolve the paraformaldehyde completely in the solution. Since the pH of PBS is around 7.4, paraformaldehyde dissolves easily in the PBS. A few drops of NaOH is added to dissolve paraformaldehyde if you are preparing a solution in water.

Step 2: Adjust the volume to 100 ml with PBS.
♦ Transfer 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 3: Filter the solution by passing through a 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 room temperature for a limited time. 

Note:
Storing 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 white precipitate upon thawing, keep the solution on the 60°C water bath until the solution becomes clear.

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