A 4% Paraformaldehyde Solution can be prepared by diluting a concentrated 16% paraformaldehyde stock solution. To prepare 100 ml of 4% paraformaldehyde solution in PBS, take 25 ml of 16% paraformaldehyde solution and add 10 ml of 10x PBS and 65 ml water. The resulting solution will have 4% paraformaldehyde in 1x PBS.
- Fixation of Cells and Tissue
- Preparation of 1N Sodium Hydroxide (NaOH) Solution by Diluting 10N Solution
Reagents and solutions
♦ 16% Paraformaldehyde stock solution (in water)
♦ 1M Sodium hydroxide (NaOH) (optional)
♦ 1M Hydrogen Chloride (HCl) (optional)
♦ 10x Phosphate-Buffered Saline (PBS)
Equipment and disposables
♦ Fume hood
♦ Conical flask / Beaker
♦ Aluminum foil / Parafilm
♦ 15 ml tubes / 50 ml tubes
4% paraformaldehyde in 1x PBS
Preparation of 100 ml of 4% paraformaldehyde solution in PBS using a 16% concentrated stock solution of paraformaldehyde
PROCEDURE: Preparation of 4% Paraformaldehyde Solution in PBS from 16% Stock Solution
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: Take 65 ml water in a 100 ml conical flask/beaker. Add 10 ml of 10x PBS and 25 ml of 16% paraformaldehyde solution and mix it properly.
If you are using the frozen stock of 16% paraformaldehyde, make sure that it is thawed completely and has no precipitate. If there is any precipitate, dissolve it by keeping it in a 60°C water bath.
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/1M NaOH.
Step 3: Filter the solution by passing it through a filter paper (Whatman No. 1 filter paper) to remove undissolved particles.
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).
Storing the solution at room temperature or 4°C will lead to the formation of formic acid in the solution.
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.