How to Make Phosphate Buffered Saline
How to Make This Immunohistochemical Staining Buffer
Phosphate buffered saline (PBS) is a buffer solution that's commonly used for immunohistochemical (IHC) staining and it's often used in biological research. PBS is a water-based salt solution containing sodium hydrogen phosphate, sodium chloride and, in some cases, potassium chloride and potassium dihydrogen phosphate.
Immunohistochemistry refers to the process of detecting antigens such as proteins in the cells of a tissue section by using the principle of antibodies binding specifically to antigens in biological tissues. Immunofluorescent staining was the first immunohistochemical staining method.
Antigens become visible when conjugated with antibodies using fluorescence dyes due to an antigen-antibody binding reaction. This process occurs when it's activated by exciting light of a specific wavelength under a fluorescent microscope.
The osmolarity and ion concentrations of the solutions match those of the human body—they're isotonic.
A Recipe for PBS
You can prepare PBS in several ways. There are multiple formulas. Some of them don't contain potassium, while others contain calcium or magnesium.
This recipe is relatively easy. It's for 10X PBS stock solution (0.1M). However, you can also make a 1X stock solution, or begin with this 10X recipe and dilute it to 1X.
The whole process takes about 10 minutes.
An option to add Tween is also provided.
What You'll Need to Make PBS Buffer
- Sodium phosphate monobasic (anhydrous)
- Sodium phosphate dibasic (anhydrous)
- Sodium chloride
- Scale and weigh boats
- Magnetic stirrer and stir bar
- A pH probe that's calibrated and appropriate solutions for adjusting pH
- 1L volumetric flask
- Tween 20 (optional)
How to Make Phosphate Buffered Saline (PBS)
Weigh 10.9g anhydrous sodium phosphate dibasic (Na2HPO4), 3.2g anhydrous sodium phosphate monobasic (NaH2PO4), and 90g sodium chloride (NaCl). Dissolve in just under 1L distilled water.
Adjust the pH to 7.4 and make the solution up to a final volume of 1L.
Dilute 10X prior to use and readjust the pH if necessary.
You can make a PBS solution containing 0.5 percent Tween 20 by adding 5mL Tween 20 to the 1L solution.
Tips for Making PBS Buffer
Store the buffer at room temperature after you've made the PBS solution.
Non-anhydrous reagents can be substituted but you'll have to recalculate the appropriate mass of each to accommodate the added water molecules.
Uses of Phosphate Buffered Saline (PBS)
Phosphate buffered saline has many uses because it's isotonic and non-toxic to most cells. It can be used to dilute substances and it's often used to rinse containers of cells. PBS can be used as a dilutent in various methods to dry biomolecules because the water molecules within it will be structured around the substance—protein, for example. It will be "dried" and immobilized to a solid surface.
The pH remains steady and consistent to prevent the destruction of cells.
The thin film of water that binds to the substance prevents denaturation or other conformational changes. Carbonate buffers can be used for the same purpose but with less effectiveness.
PBS can also be used to take a reference spectrum when measuring the protein adsorption in ellipsometry.