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One for All: the Universal Buffer

We are delighted to present our innovative and patent-pending R-Universal buffer for antigen unmasking/epitope recovery on formalin-fixed, paraffin-embedded sections. The "R" in R-Universal represents Retriever, emphasizing its compatibility with the 2100 Retriever device. Extensive testing of this buffer has been conducted in pathology labs throughout the UK, ensuring its efficacy and reliability. We are excited to make it available not only to Retriever users but also to researchers working with routine tissue material

 

The underlying processes of heat-induced epitope recovery are not entirely clear, despite various claims made in the field [1]. Heat, whether applied through a microwave, pressure cooker, or water bath, leads to protein denaturation, which is believed to expose linear peptide epitopes on the protein, thus improving their accessibility [2]. However, the pH of the recovery buffer plays a critical role in achieving the desired staining outcome with a specific antibody. If the pH of the buffer is not suitable for a particular antibody, it can result in unexpected staining patterns or even no staining at all, often leading to non-specific staining [3]. Consequently, a range of buffers is recommended, with each designed for a specific epitope, such as Citrate, EDTA, Tris at different pH levels (e.g., pH 3.5, pH 6, pH 8, pH 9.5, pH 10, etc.) [4]. It is also worth noting that while Tris-based buffers may work well in the microwave, their performance in pressure-cooker types of processing units is typically suboptimal.

During fixation and drying, calcium ions are known to precipitate in the tissue and can impact staining outcomes. Furthermore, formalin, commonly used for fixation, induces cross-linking between proteins and other molecules within the tissue. Formaldehyde primarily reacts with ε-amino groups of lysine residues (reversibly), while secondary reactions form irreversible cross-links between amino residues, amino acids, and DNA [6,7]. The proposed use of citraconic anhydride for epitope recovery is based on its reversible reaction with modified ε-amino groups, resulting in improved antigen unmasking [8]. However, it should be noted that citraconic anhydride is toxic and requires lengthy epitope recovery at high temperatures (+950°C). Additionally, in our studies (unpublished), we have observed weak reactivity of certain antibodies, such as those targeting transcription factors like TTF-1, with sections recovered using citraconic anhydride.

Lastly, certain epitopes, such as those found on epithelial markers Ep-CAM, GFAP, and others, require proteolytic processing for successful epitope recovery

Additionally, the quality of epitope recovery is significantly influenced by the quality of fixation and processing. Factors such as the duration of fixation, thorough washing to remove fixative residues, and the temperature of the paraffin used can all impact the effectiveness of retrieval. In situations where large series of stainings are performed on collections from archives, which have been formed over time, there is a risk of variations in the efficiency of epitope retrieval. We have had clients who, prior to adopting the Retriever, experienced false negatives in up to 30% of cases, even for well-established markers such as the estrogen receptor. This highlights the potential magnitude of the problem when working with less routine antibodies.

In the image provided, staining results are displayed using three standard antibodies from Dako: Cyclin D1, Lambda chain, and GFAP. Each of these antibodies typically requires a specific buffer (Low, High, or protease treatment) to achieve proper epitope recovery and avoid non-specific staining.

In the left panel, staining is performed on sections processed with the recommended buffer for each antibody. In the last panel, staining is performed using the R-Universal buffer. Notably, there is a significant improvement in the discrimination between positive and negative cells, as well as stronger staining observed for Cyclin D1 and Lambda chain when using the R-Universal buffer.

This demonstrates the enhanced performance and benefits of the R-Universal buffer in achieving better staining results, improved discrimination between positive and negative cells, and stronger overall staining intensity when compared to using the specific recommended buffers for individual antibodies.

We are confident that with the 2100 Retriever and our new R-Universal buffer, we have achieved an exceptional solution for epitope recovery. The specially designed heating-cooling program in the Retriever was specifically developed to reshape denatured epitopes, allowing for proper refolding. The R-Universal buffer goes a step further by effectively removing formaldehyde-formed protein cross-links and calcium ions, without the need for toxic agents such as Citraconic anhydride.

As a result, you can now perform epitope recovery for practically all epitopes using a single buffer. This offers several advantages:

  1. Treatment of all sections in one buffer, minimizing wastage when dealing with multiple small series of sections that require different buffers.

  2. Replacement of various high, low, or other specific buffers, while still achieving successful epitope recovery.

  3. Recovery of epitopes that typically require protease treatment (e.g., Trypsin, Proteinase K) can now be achieved with the R-Universal buffer.

  4. Use of antibodies that were previously untested or showed limited success on formalin-fixed sections.

  5. Performance of immunofluorescence staining on formalin-fixed sections, as the autofluorescence caused by excess unwashed formaldehyde will be eliminated during section processing.

  6. Conducting multicolor immunofluorescence staining on sections, even when using antibodies that traditionally require epitope recovery in different buffers.

  7. Obtaining stronger staining results, surpassing other methods of epitope recovery. This provides greater confidence in identifying true negative results, without concerns about inadequate epitope recovery.

In summary, you can enjoy the convenience of performing immunohistochemistry on frozen sections while preserving excellent tissue morphology associated with formalin-fixed samples

Product data sheet

References

1. T.Mellows, S.Litvinov, G.Thomas, A novel universal epitope recovery buffer for formalin-fixed sections: (in preparation)

 

2. Leong, T.Y.-M., and Leong, A.S.-Y. (2007). How does antigen retrieval work? Adv Anat Pathol 14, 129–131.

 

3. Fowler, C.B., Evers, D.L., O’Leary, T.J., and Mason, J.T. (2011). Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity. J. Histochem. Cytochem. 59, 366–381.

 

4. Emoto, K., Yamashita, S., and Okada, Y. (2005). Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens? J. Histochem. Cytochem. 53, 1311–1321.

 

5. Shi, S.-R., Shi, Y., and Taylor, C.R. (2011). Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades. J. Histochem. Cytochem. 59, 13–32.

 

6.  Syrbu, S.I., and Cohen, M.B. (2011). An enhanced antigen-retrieval protocol for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues. Methods Mol. Biol. 717, 101–110.

 

7. Siomin, Y.A., Simonov, V.V., and Poverenny, A.M. (1973). The reaction of formaldehyde with deoxynucleotides and DNA in the presence of amino acids and lysine-rich histone. Biochim. Biophys. Acta 331, 27–32.

 

8. Namimatsu, S., Ghazizadeh, M., and Sugisaki, Y. (2005). Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method. J. Histochem. Cytochem. 53, 3–11.

Gallery of published Images

(epitopes were recovered using R-Universal)

Konger, R.L., Derr-Yellin, E., Ermatov, N., Ren, L., and Sahu, R.P. (2019). The PPARγ Agonist Rosiglitazone Suppresses Syngeneic Mouse SCC (Squamous Cell Carcinoma) Tumor Growth through an Immune-Mediated Mechanism. Molecules 24.

 

Rupp, C., Aakula, A., Isomursu, A., Erickson, A., Kauko, O., Shah, P., Padzik, A., Kaur, A., Li, S.-P., Pokharel, Y.R., et al. (2019). PP2A inhibitor PME-1 suppresses anoikis, and is associated with therapy relapse of PTEN-deficient prostate cancers. BioRxiv 581660.

 

Störmann, P., Becker, N., Vollrath, J.T., Köhler, K., Janicova, A., Wutzler, S., Hildebrand, F., Marzi, I., and Relja, B. (2019). Early Local Inhibition of Club Cell Protein 16 Following Chest Trauma Reduces Late Sepsis-Induced Acute Lung Injury. Journal of Clinical Medicine 8, 896.

 

Wagner, N., Dieteren, S., Franz, N., Köhler, K., Perl, M., Marzi, I., and Relja, B. (2019). Alcohol‑induced attenuation of post‑traumatic inflammation is not necessarily liver‑protective following trauma/hemorrhage. International Journal of Molecular Medicine 44, 1127–1138.

 

Bennett, C.L., Dastidar, S.G., Ling, S.-C., Malik, B., Ashe, T., Wadhwa, M., Miller, D.B., Lee, C., Mitchell, M.B., van Es, M.A., et al. (2018). Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients. Acta Neuropathol 136, 425–443.

Doster, R.S., Sutton, J.A., Rogers, L.M., Aronoff, D.M., and Gaddy, J.A. (2018). Streptococcus agalactiae Induces Placental Macrophages To Release Extracellular Traps Loaded with Tissue Remodeling Enzymes via an Oxidative Burst-Dependent Mechanism. MBio 9, e02084-18.

 

Heinzelmann, K., Lehmann, M., Gerckens, M., Noskovičová, N., Frankenberger, M., Lindner, M., Hatz, R., Behr, J., Hilgendorff, A., Königshoff, M., et al. (2018). Cell-surface phenotyping identifies CD36 and CD97 as novel markers of fibroblast quiescence in lung fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology

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Maskari, R.A., Hardege, I., Cleary, S., Figg, N., Li, Y., Siew, K., Khir, A., Yu, Y., Liu, P., Wilkinson, I., et al. (2018). Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness. European Journal of Human Genetics 26, 1648.

 

Brinkmann, V., Abu Abed, U., Goosmann, C., and Zychlinsky, A. (2016).

Immunodetection of NETs in Paraffin-Embedded Tissue. Front Immunol 7.

 

Schumacher, F.-R., Siew, K., Zhang, J., Johnson, C., Wood, N., Cleary, S.E.,

Al Maskari, R.S., Ferryman, J.T., Hardege, I., Yasmin, et al. (2015). Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia. EMBO Molecular Medicine 7, 1285–1306.

 

Zhang, J., Siew, K., Macartney, T., O’Shaughnessy, K.M., and Alessi, D.R. (2015). Critical role of the SPAK protein kinase CCT domain in controlling blood pressure. Hum. Mol. Genet. ddv185.

Published Work Using R-Universal
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