Applications for Sodium Citrate Buffer

Why is Sodium Citrate used as a Buffer?

Sodium citrate buffer, also known as citrate buffer, is a common reagent used for antigen retrieval in immunohistochemistry. Immunohistochemistry (IHC) is a technique that enables high-quality visualization of proteins in a variety of tissues. IHC is a multi-step process that comprises fixation, embedding, and sectioning of a target tissue followed by incubation with an antibody targeting a protein of interest.

Once the tissue is fixed, embedded in paraffin, and sectioned on slides, researchers can utilize antibodies to target and identify their protein of interest. However, fixation and embedding of tissues, often with 10% formalin and paraffin respectively, initiates cross-linking of proteins that can mask antigen epitopes. Antigen masking may reduce the sensitivity of antibodies and decrease the detection of certain proteins of interest. A process known as antigen retrieval breaks the bonds between formalin and proteins, removing the effects of cross-linking, thereby enabling better detection of proteins. Sodium citrate buffer is highly used in the process of antigen retrieval, specifically for heat-induced antigen retrieval. Boster Bio offers a simple-to-use Sodium Citrate Buffer (Catalog# AR0024) for heat-induced antigen retrieval to remove the effects of antigen masking in your IHC experiments.

What is Antigen Retrieval?

Antigen retrieval is a process of breaking the bonds from cross-linking, enabling proteins to interact with antibodies during immunohistochemistry. The purpose of antigen retrieval in immunohistochemistry is to unmask antigens in fixed tissues, which can significantly improve the quality of staining and detection of proteins. While optimization of this method may add additional steps to an already multi-step, complex procedure, antigen retrieval has improved IHC and vastly increased the ability of researchers to visualize proteins that were otherwise elusive. Antigen retrieval occurs typically by two main methods—heat-induced antigen retrieval (HIER) or protease-induced antigen retrieval (PIER).

Sodium Citrate Buffer in Heat-induced antigen retrieval (HIER) vs. Protease-induced antigen retrieval (PIER):

How is Sodium Citrate Buffer used in heat-induced antigen retrieval (HIER)?

Heat-induced antigen retrieval, also known as HIER, was first introduced as a method in the early 1990s when immunohistochemical slides were dipped in boiling water. Although heat typically denatures proteins and eliminates common epitopes, submersing these samples in boiling water removed antigen masking by breaking the cross-linking bonds from fixation. Since then, a variety of buffers and methods have been optimized for heat-induced antigen retrieval.

One of the most common solutions used in HIER is Sodium Citrate buffer (pH 6.0). Studies have noted that the use of citrate buffer in antigen retrieval typically provides enormous benefits and allows for excellent staining. Heat-induced antigen retrieval solutions, like sodium citrate buffer, are heated prior to use. IHC slides are incubated in the buffer for about 15-20 minutes while boiling, though if lower temperatures are required the time can be increased. After using heat-induced or protease-induced heat antigen reagents, slides are removed from heat, allowed to cool to room temperature, then washed in PBS.

IHC antigen retrieval protocols are often very simple and can improve the quality of your staining and dramatically increase the detection of desired proteins. It is recommended to optimize antigen retrieval conditions for each new antibody or to search for already optimized conditions.

Why is sodium citrate buffer used instead of EDTA?

Other commonly used buffers include EDTA buffer (pH 8.0) and Tris-EDTA (pH 9.0). Both Sodium Citrate Buffer and EDTA buffers are used in a similar manner. IHC slides incubate in the heated buffer for a length of time. While EDTA buffers work well in a number of conditions, these reagents tend to be much more aggressive and can damage antigens and fixed tissue morphology. For more sensitive antigens or when tissue structure may be compromised easily, it is recommended to try antigen retrieval with a more sensitive reagent, like citrate buffer. For your IHC experiments, Boster Bio offers a well-established, highly cited Sodium Citrate Buffer (Catalog# AR0024) to eliminate antigen masking in fixed tissue sections while preserving overall tissue structure.

How does protease-induced antigen retrieval (PIER) compare to sodium citrate buffer in heat-induced antigen retrieval (HIER)?

Protease-induced antigen retrieval, or PIER, is a similar method that removes cross-linking due to fixation. However, PIER utilizes proteases to eliminate these cross-links rather than heat. While protease-induced antigen retrieval is useful in a variety of conditions and can be an optimal antigen retrieval reagent, it requires more optimization of conditions including temperature, protease concentration, incubation time, and fixation time compared to sodium citrate buffer. Additionally, proteases also tend to alter tissue morphology in fixed samples.

Common proteases used for antigen retrieval include trypsin (0.05%), pepsin (0.5%), and proteinase K (20μg/mL). Both trypsin and pepsin are pre-heated to about 37 °C, then IHC slides are incubated in the solution for about 10-20 minutes (less time is typically required for pepsin digestion). Proteinase K works much faster and can be used at room temperature, with IHC slide incubation only being approximately 2-4 minutes. After incubation with any protease, IHC slides are washed with PBS or PBS-Tween, depending on the protocol. It is important to remember that the optimal conditions for antigen retrieval are often dependent on individual antigens, primary antibodies, and tissue type.

How do you make a sodium citrate buffer?

Sodium citrate buffer is an exceedingly easy buffer to prepare. Typical preparation includes dissolving trisodium citrate in distilled water, then bringing the pH to 6.0 with hydrochloric acid. For ease of use, Boster Bio offers citrate buffer in powder form (Catalog# AR0024) that can be dissolved in water to prepare up to a 2 Liter solution. Once made, the citrate buffer can be stored at room temperature for up to one year.

References

  • Cheng, X., Qin, L., Deng, L., Zhu, X., Li, Y., Wu, X., & Zheng, Y. (2021). SNX-2112 Induces Apoptosis and Inhibits Proliferation, Invasion, and Migration of Non-Small Cell Lung Cancer by Downregulating Epithelial-Mesenchymal Transition via the Wnt/β-Catenin Signaling Pathway. Journal of Cancer, 12(19), 5825–5837. https://doi.org/10.7150/jca.56640
  • Delcambre, G. H., Liu, J., Herrington, J. M., Vallario, K., & Long, M. T. (2016). Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue. PeerJ, 4, e1601. https://doi.org/10.7717/peerj.1601
  • Dunkenberger, L., & Del Valle, L. (2022). Antigen Retrieval and Signal Amplification. In L. Del Valle (Ed.), Immunohistochemistry and Immunocytochemistry: Methods and Protocols (pp. 65–74). Springer US. https://doi.org/10.1007/978-1-0716-1948-3_5
  • Sodium citrate buffer. (2009). Cold Spring Harbor Protocols, 2009(12), pdb.rec12040. https://doi.org/10.1101/pdb.rec12040
  • Sompuram, S. R., Vani, K., Schaedle, A. K., Balasubramanian, A., & Bogen, S. A. (2019). Selecting an Optimal Positive IHC Control for Verifying Antigen Retrieval. Journal of Histochemistry and Cytochemistry, 67(4), 275–289. https://doi.org/10.1369/0022155418824092

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