Citrate buffer is a solution commonly used in
histological and immunohistochemical techniques to retrieve antigens that have been masked by formalin fixation and paraffin embedding. The buffer is composed of citric acid and its conjugate base, sodium citrate, and maintains a stable pH, typically around 6.0.
In histology, tissues are often fixed using
formalin to preserve cellular structures. However, this fixation process can mask or alter antigenic sites, making it challenging for antibodies to bind during immunohistochemical staining. Citrate buffer helps to unmask or retrieve these antigens, ensuring accurate and reliable
immunostaining.
Citrate buffer works through a process known as Heat-Induced Epitope Retrieval (HIER). This involves heating the tissue sections in citrate buffer, which helps to break the cross-links formed during formalin fixation. The heating process can be done using a microwave, pressure cooker, or water bath. This step is crucial for improving the binding efficiency of antibodies to their respective antigens.
Citrate buffer is primarily used in immunohistochemistry (IHC) and
immunocytochemistry to enhance antigen-antibody interactions. It is also used in other histological techniques to improve staining quality and tissue morphology. Additionally, citrate buffer is sometimes employed in enzyme histochemistry and in situ hybridization protocols.
To prepare a 10 mM citrate buffer solution at pH 6.0, dissolve 2.1 g of citric acid (C6H8O7) and 2.94 g of sodium citrate (C6H5O7Na3ยท2H2O) in 1 liter of distilled water. Adjust the pH to 6.0 using either citric acid or sodium citrate. Filter the solution to remove any particulate matter and store it at room temperature.
Citrate buffer offers several advantages in histological applications:
1. Effective Antigen Retrieval: It efficiently unmask antigens, leading to improved antibody binding.
2. Versatility: Suitable for use in various tissue types and with different antibodies.
3. Cost-Effective: Inexpensive and easy to prepare.
4. Compatibility: Works well with other buffers and reagents used in histology.
While citrate buffer is highly effective, it may not be suitable for all types of antigens or tissues. Some antigens may require different retrieval buffers or methods. Additionally, excessive heating during antigen retrieval can sometimes damage the tissue morphology, so optimization of retrieval conditions is essential.
Conclusion
Citrate buffer plays a critical role in the field of histology by enhancing antigen retrieval during immunohistochemical staining. Its effectiveness, ease of preparation, and cost-efficiency make it a popular choice among researchers. However, as with any technique, it is important to optimize conditions for each specific application to achieve the best results.
