How to prepare a 4% Paraformaldehyde (PFA) Solution in PBS

Caution!

Paraformaldehyde is toxic. Please read the MSDS before working with this chemical. Avoid contact with skin, eyes, or mucous membranes. Avoid breathing the powder during measuring and preparation. Gloves and safety glasses should be worn and solutions made inside a fume hood.


Function

Paraformaldehyde is primarily used in biological and biomedical research for fixing cells and tissues. When dissolved in a buffer like PBS (phosphate-buffered saline) to make a 4% solution, its main function is to crosslink proteins within cells and tissues. This fixation process preserves cellular morphology and prevents degradation, enabling various microscopic techniques such as immunostaining and microscopy for detailed analysis of cellular structures and protein localization. Additionally, paraformaldehyde fixation stabilizes antigens and prevents autolysis and putrefaction, making it essential for accurate scientific research and histological studies.

DEPC functions as a natural inhibitor of RNAases, effectively suppressing their activity in ex vivo tissue, thus preserving RNA integrity. Tissues fixed in formaldehyde solution containing DEPC are suitable for both immunohistochemistry and in situ hybridization, whereas those lacking DEPC are generally only suitable for immunohistochemistry. Boster offers an affordable 4% Paraformaldehyde (PFA) Solution In PBS (With DEPC) (Catalog# AR1068) that has been validated and cited in many publications.


Protocol

The protocols below describe the technique for generating a 4% paraformaldehyde solution in PBS with or without DEPC.

Please read the protocol in its entirety before starting。

Required Reagents and Materials

4% paraformaldehyde solution in PBS4% paraformaldehyde solution in PBS with DEPC
Deionized H2ODEPC
Dilute HClDeionized H2O
NaOH (1 N)Dilute HCl
Paraformaldehyde powderNaOH (1 N)
1X PBSParaformaldehyde powder
1X PBS

Materials

Filter units

Glassware and stir bar

Gloves and eye protection

Hot plate with a magnetic stirrer

Thermometer

Ventilated hood


Procedure

Here we are describing steps for 4% Paraformaldehyde of the 1L solution. The same trend follows for other volumes as well.


4% paraformaldehyde solution in PBS

1. Add 800 mL of 1X PBS to a glass beaker on a stir plate in a ventilated hood. Heat while stirring to approximately 60 °C. Take care that the solution does not boil, use glass thermometer to monitor. (Temperatures above 60 °C will denature the paraformaldehyde, making it useless as a fixative.)
2. Add 40 g of paraformaldehyde powder to the heated PBS solution.
3. The powder will not immediately dissolve into solution. Slowly raise the pH by adding 1 N NaOH dropwise from a pipette until the solution clears.
4. Once the paraformaldehyde is dissolved, remove from heat and cool to room temperature and filter.
5. Adjust the volume of the solution to 1 L with 1X PBS.
6. Adjust the pH with small amounts of dilute HCl to 7.2-7.6.
7. The solution can be aliquoted and stored at -20 °C.


4% paraformaldehyde solution in PBS with DEPC

1. Add DEPC to 1X PBS at a final concentration of 0.1%.
2. Autoclave the DEPC-treated PBS to inactivate DEPC (autoclaving is necessary to remove residual DEPC that can inhibit cellular functions).
3. Add 800 mL of DEPC-treated PBS to a glass beaker on a stir plate in a ventilated hood. Heat while stirring to approximately 60 °C. Take care that the solution does not boil, use glass thermometer to monitor.
4. Add 40 g of paraformaldehyde powder to the heated PBS solution.
5. The powder will not immediately dissolve into solution. Slowly raise the pH by adding 1 N NaOH dropwise from a pipette until the solution clears.
6. Once the paraformaldehyde is dissolved, remove from heat and cool to room temperature and filter.
7. Adjust the volume of the solution to 1 L with DEPC-treated PBS.
8. Adjust the pH with small amounts of dilute HCl to 7.2-7.6.
9. The solution can be aliquoted and stored at -20 °C.



Difference Between Formalin, Formaldehyde And ParaformaldehydeFormaldehyde:

Formalin, formaldehyde, and paraformaldehyde are related compounds used in biological and medical sciences, each with specific properties and applications. Here is a breakdown of their differences:


Formaldehyde:

Chemical Structure: Formaldehyde (CH₂ is the simplest form of aldehyde, consisting of a carbon atom bonded to two hydrogen atoms and an oxygen atom (H-C=O).

Physical State: Formaldehyde is typically encountered as a gas at room temperature and under normal atmospheric pressure. However, it is often sold and used as an aqueous solution known as formalin.

Use: It is a highly reactive compound and a potent fixative. In biological and medical contexts, it is used primarily as a component of formalin solutions for tissue fixation.


Formalin:

Composition: Formalin is an aqueous solution of formaldehyde gas dissolved in water, typically containing about 37-40% formaldehyde by weight (approximately 10-15% formaldehyde by volume).

Function: Formalin is used extensively as a fixative in histology and pathology to preserve tissues for microscopy and other analyses. It crosslinks proteins and nucleic acids, preventing their degradation and maintaining tissue structure.


Paraformaldehyde:

Chemical Structure: Paraformaldehyde (PFA) is a polymer of formaldehyde, consisting of a linear chain of repeating formaldehyde units (-(CH₂O)ₙ-).

Physical State: Paraformaldehyde is a solid at room temperature and must be depolymerized into formaldehyde for use in aqueous solutions.

Preparation and Use: It is typically dissolved in a buffer (such as PBS) to create a solution used for tissue fixation and cell permeabilization in biological research. Once dissolved, paraformaldehyde releases formaldehyde gas, which then acts as a fixative.