How to Make Phosphate Buffered Saline (PBS) for IHC

PBS is a commonly used buffer for immunohistochemical staining

Immunohistochemical staining
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PBS (phosphate buffered saline) is a commonly used buffer for immunohistochemical staining and is commonly used in biological research. The osmolarity and ion concentrations of the solutions match those of the human body (isotonic).

PBS is a water-based salt solution containing sodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and potassium dihydrogen phosphate.

Immunofluorescent Staining for IHC

Immunofluorescent staining is the first immunohistochemical (IHC) staining method. With fundamentality of antigen-antibody binding reaction, antigens are visualized by fluorescence dyes conjugated with antibodies when being activated by exciting light of specific wavelength under fluorescence microscope. Immunohistochemistry refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.

Uses of Phosphate Buffered Saline (PBS)

Phosphate buffered saline has many uses because it is isotonic and nontoxic to most cells. It can be used to dilute substances. It is used to rinse containers containing cells. PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be ‘dried’ and immobilized to a solid surface.

The thin film of water that binds to the substance prevents denaturation or other conformational changes. Carbonate buffers may be used for the same purpose but with less effectiveness. PBS can be used to take a reference spectrum when measuring the protein adsorption in ellipsometry.

Additives can be used to add function.

For example, PBS with EDTA is also used to disengage attached and clumped cells. Divalent metals such as zinc, however, cannot be added as this will result in precipitation. 

Preparation of PBS

There are many different ways to prepare PBS. Some formulations do not contain potassium, while others contain calcium or magnesium. The buffer recipe provided here, which is relatively easy, is for 10X PBS stock solution (0.1M). An option for added Tween is also provided. The whole process takes about 10 minutes.

How to Make Phosphate Buffered Saline (PBS) 

  1. Weigh the following: 10.9g anhydrous sodium phosphate dibasic (Na2HPO4), 3.2g anhydrous sodium phosphate monobasic (NaH2PO4), and 90g sodium chloride (NaCl).
  2. Dissolve all of the above in just under 1L distilled water.
  3. Adjust the pH to 7.4.
  4. Make the solution up to a final volume of 1L.
  5. (optional) For a solution containing 0.5% Tween 20, add 5mL Tween 20 to the 1L solution.
  6. Dilute 10X prior to use and readjust pH if necessary.

Tips for Making PBS Buffer:

  • Store at room temperature.
  • Non-anhydrous reagents may be substituted but you'll have to recalculate the appropriate mass of each to use, making accommodation for the added water molecules.

What You Need to Make PBS Buffer:

  • Sodium phosphate monobasic (anhydrous)
  • Sodium phosphate dibasic (anhydrous)
  • Sodium chloride
  • Scale and weigh boats
  • Magnetic stirrer and stir bar
  • pH probe, calibrated, and appropriate solutions for adjusting pH
  • 1L volumetric flask
  • Tween 20 (optional)

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