One for All: the Universal Buffer

We proudly present our novel, patent-pending R-Universal (R stands for Retriever) buffer for antigen unmasking/epitope recovery on formalin-fixed, paraffin embedded sections[1]. Being specifically developed for 2100 Retriever, it was thoroughly tested in pathology labs across the UK, and is now available for all users of Retriever and other researchers who work with routine tissue material.

 

The processes that are really lying behind heat-induced epitope recovery are not really clear, irrespective of what some may claim [1]. First of all, heat (microwave or pressure cooker, or just a water bath) leads to denaturation of the protein, which is believed to result in better exposure of linear peptide epitopes on the protein [2]. However, if the pH of the recovery buffer does not “suit” a particular individual antibody, the resulting staining will be far from expected (a totally different pattern may be observed, or no staining at all), usually non-specific [3]. Thus, so many buffers are recommended, usually – one particular for a particular epitope: Citrate, EDTA, Tris, pH 3. 5, pH 6, pH8, pH 9.5, pH 10, etc [4]. Moreover, i.e. Tris –based buffers work for some antibodies in the microwave, but usually perform poorly in pressure-cooker types of  processing units.   

Adding some protein-denaturating agents to the buffer (such as SDS [5] or Guanindin, as we used in our previous  U buffers) may  improve reactivity of some antibodies with recovered section, but may destroy the epitope for others. Attempts to use nonionic detergents for better recovery, again, showed recovery of some epitopes, and created a lot of non-specific staining for others. It was also believed that during fixation and drying, Ca++ ions precipitate in tissue and affect the staining. And, finally, formalin used for fixation produces a number of cross-links between proteins and other molecules in the tissue. Primary reaction of formaldehyde in tissue is with ε-amino groups of lysines (considerate to be reversible); secondary reactions form cross-links between amino residues, and amino acids and DNA[6,7], and these reactions are considered irreversible. Proposed use of citraconic anhydride for epitope recovery [8] is based on agent’s reversible reaction with modified ε-amino group, and indeed improves antigen unmasking. Unfortunately, citraconic anhydride is toxic, and requires epitope recovery for a long time at +950. In our studies, we have also found that some antibodies react weakly with sections recovered by citraconic anhydride (unpublished), i.e. some antibodies to transcription factors, in particular TTF-1.

Finally, some epitopes (i.e. most of the epitopes on epithelial markers Ep-CAM, GFAP and some others) require proteolytic processing.

Moreover, the quality of recovery greatly depends on the quality of fixation and processing, in particular – length of fixation, quality of washing from the fixative, and the temperature of paraffin used. When one performs large series of stainings on collections from archives, formed over some time, there is a risk of differences in efficacy of retrieval (we have clients who, before they switched to Retriever, had up to 30% of false-negatives, even for such established marker as oestrogen receptor; imagine the possible size of the problem when less routine antibody is used).

On the image you see staining with  three standard antibodies from Dako, to Cyclin D1, Lambda chain, and GFAP. Each of these require individual buffer (Low, High, or protease treatment) for a proper epitope recovery to avoid a non-specific staining produced by the respective mAb. Left panel shows staining on sections processed with recommended buffer, on the ladt panel - with R-universal buffer. Please not a much better discrimination between positive and negative cells (and stronger staining) for CyclinD1 and Lambda chain.

We believe that we have achieved an excellent solution for epitope recovery with 2100 Retiever, and the new R-Universal buffer. The specially designed program of heating-cooling in Retriever was from the very beginning  created for the best results in reshaping the denatured epitopes (allowing their proper refolding). But the new Retrieval buffer combines the removal of formaldehyde-formed protein cross-links with the removal of Ca++. And no toxic agent, such as Citraconic anhydride, is used.

 

As a result, you can perform recovery of practically all epitopes in one buffer.

 

Which allows you:

 

  • Treat all sections in one buffer, so no buffer is wasted, when you have to treat several small series of sections in different buffers

  • To use one buffer not only instead of different High, Low, etc buffers, but also to recover epitopes that normally require protease (Trypsin, Proteinase K) treatment.

  • To use practically any antibody previously not tested (or unsuccessfully tested) on formalin-fixed sections.

  • To perform IF staining formalin-fixed on sections, as autofluorescence, even produced by excess of unwashed formaldehyde, will go away during the section processing.

  • To perform multicolour IF on sections, even when using antibodies that normally require epitope recovery in different buffers

  • To get the strongest staining, superior to any other methods for epitope recovery. You will have more confidence that what you see as negative -  is indeed negative, and not a result of poor epitope recovery.

 

In other words, you may have all the conveniences of IHC on frozen sections, while having excellent tissue morphology of formalin-fixed.

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.

PME-1 protein expression. Microarray
PME-1 protein expression. Microarray

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.

Costainings of CDCP1 (red) and -SMA
Costainings of CDCP1 (red) and -SMA

Noskovičová, N., Heinzelmann, K., Burgstaller, G., Behr, J., and Eickelberg, O. (2018). Cub domain-containing protein 1 negatively regulates TGF-β signaling and myofibroblast differentiation. American Journal of Physiology-Lung Cellular and Molecular Physiology 314, L695–L707.

pNCC in distal tubule (tNCC)
pNCC in distal tubule (tNCC)

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.

PME-1 protein expression. Microarray
PME-1 protein expression. Microarray

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.

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

To see the full reference and the original article, please click on image and follow the link