Paraformaldehyde 4 1000mL



Final concentration


40 g



500 mL

0.2 M Phosphate buffer pH 7.4

500 mL

0.1 M, pH 7.4

In fume hood:

1. Add 40 g of paraformaldehyde to 500 mL of dH2O.

2. Heat to 60°C (do not exceed this temperature).

3. Stir until dissolved.

4. Add a few drops of 1N NaOH until solution is clear. (Will not completely dissolve or clear without the addition of NaOH.)

5. Filter (0.2 or 0.45 |m) and add 500 mL of 0.2 M phosphate buffer, pH to 7.4 (recheck pH and adjust if necessary).

6. Store at 4°C up to one week (alternatively store aliquots at -20°C).

Important notes about fixative preparation and storage


■ We recommend that paraformaldehyde be prepared fresh whenever possible. It will make a noticeable difference in the quality of immunofluorescence.

■ Solubilization of paraformaldehyde powder is often accomplished with heat and strong base. Take care not to heat above 55-60°C and add just enough base to dissolve the paraformaldehyde. Under high heat or high pH, paraformaldehyde will iso-convert to formaldehyde which degrades rapidly to formic acid and water.

■ If the solution temperature goes over 65°C during preparation, do not use it as it will produce a strong autofluorescence in cells or tissues.

Formaldehyde 37%

■ Storage of 37% formaldehyde over several months results in degradation to formic acid and water.

■ Old formaldehyde stocks should be disposed of every 12-24 months.

Buffered formalin 10%

■ 10% "buffered formalin": pH will drift due to degradation of fomaldehyde to formic acid.

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  • hayden
    How to make 4% paraformaldehyde from 37% stock?
    8 years ago

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