https://doi.org/10.4081/ejh.2023.3812
Management of autofluorescence in formaldehyde-fixed myocardium: choosing the right treatment
Submitted: 21 June 2023
Accepted: 28 August 2023
Published: 2 October 2023
Accepted: 28 August 2023
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All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Authors
Autofluorescence (AF) poses challenges for detecting proteins of interest in situ when employing immunofluorescence (IF) microscopy. This interference is particularly pronounced in strongly autofluorescent tissues such as myocardium, where tissue AF can be comparable to IF. Although various histochemical methods have been developed to achieve effective AF suppression in different types of tissue, their applications on myocardial samples have not been well validated. Due to inconsistency across different autofluorescent structures in sometypes of tissue, it is unclear if these methods can effectively suppress AF across all autofluorescent structures within the myocardium. Here, we quantitatively evaluated the performance of several commonly used quenching treatments on formaldehyde-fixed myocardial samples, including 0.3 M glycine, 0.3% Sudan Black B (SBB), 0.1% and 1% sodium borohydride (NaBH4), TrueVIEW® and TrueBlack®. We further assessed their quenching performance by employing the pre-treatment and post-treatment protocols, designed to cover two common IF staining scenarios where buffers contained detergents or not. The results suggest that SBB and TrueBlack® outperform other reagents in AF suppression on formaldehyde-fixed myocardial samples in both protocols. Furthermore, we inspected the quenching performance of SBB and TrueBlack® on major autofluorescent myocardial structures and evaluated their influence on IF imaging. The results suggest that SBB outperforms TrueBlack® in quenching major autofluorescent structures, while TrueBlack® excels in preserving IF labeling signal. Surprisingly, we found the treatment of NaBH4 increased AF signal and enhanced the AF contrast of major autofluorescent structures. This finding suggests that NaBH4 has the potential to act as an AF enhancer and may facilitate the interpretation of myocardial structures without the need for counterstaining.
Bastiaens PIH, Pepperkok R. Observing proteins in their natural habitat: The living cell. Trends Biochem Sci 2000;25:631-7.
DOI: https://doi.org/10.1016/S0968-0004(00)01714-X
Yuan P, Condello C, Keene CD, Wang Y, Bird TD, Paul SM, et al. TREM2 haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy. Neuron 2016;90:724-39.
DOI: https://doi.org/10.1016/j.neuron.2016.05.003
Shi X, Garcia G, Van De Weghe JC, McGorty R, Pazour GJ, Doherty D, et al. Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome. Nat Cell Biol 2017;19:1178-88.
DOI: https://doi.org/10.1038/ncb3599
Wojnilowicz M, Glab A, Bertucci A, Caruso F, Cavalieri F. Super-resolution imaging of proton sponge-triggered rupture of endosomes and cytosolic release of small interfering RNA. ACS Nano 2019;13:187-202.
DOI: https://doi.org/10.1021/acsnano.8b05151
Degors IMS, Wang C, Rehman ZU, Zuhorn IS. Carriers break barriers in drug delivery: endocytosis and endosomal escape of gene delivery vectors. Acc Chem Res 2019;52:1750-60.
DOI: https://doi.org/10.1021/acs.accounts.9b00177
Wagnieres GA, Star WM, Wilson BC. In vivo fluorescence spectroscopy and imaging for oncological applications. Photochem Photobiol 1998;68:603-32.
DOI: https://doi.org/10.1111/j.1751-1097.1998.tb02521.x
Corrodi HR, Jonsson G. The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines a review on the methodology. J Histochem Cytochem 1967;15:65-78.
DOI: https://doi.org/10.1177/15.2.65
Ghasemi F, Parvin P, Lotfi M. Laser-induced fluorescence spectroscopy for diagnosis of cancerous tissue based on the fluorescence properties of formaldehyde. Laser Phys Lett 2019;16:35601.
DOI: https://doi.org/10.1088/1612-202X/aaf89d
Clancy B, Cauller LJ. Reduction of background autofluorescence in brain sections following immersion in sodium borohydride. J Neurosci Methods 1998;83:97-102.
DOI: https://doi.org/10.1016/S0165-0270(98)00066-1
Davis AS, Richter A, Becker S, Moyer JE, Sandouk A, Skinner J, et al. Characterizing and diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. J Histochem Cytochem 2014;62:405-23.
DOI: https://doi.org/10.1369/0022155414531549
Lillie RD, Pizzolato P. Histochemical use of borohydrides as aldehyde blocking reagents. Stain Technol 1972;47:13-6.
DOI: https://doi.org/10.3109/10520297209116528
Baschong W, Suetterlin R, Laeng RH. Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM). J Histochem Cytochem 2001;49:1565-71.
DOI: https://doi.org/10.1177/002215540104901210
Romijn HJ, van Uum JFM, Breedijk I, Emmering J, Radu I, Pool CW. Double immunolabeling of neuropeptides in the human hypothalamus as analyzed by confocal laser scanning fluorescence microscopy. J Histochem Cytochem 1999;47:229-35.
DOI: https://doi.org/10.1177/002215549904700211
Oliveira VC, Carrara RC V, Simoes DLC, Saggioro FP, Carlotti CGJ, Covas DT, et al. Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections. Histol Histopathol 2010;25:1017-24.
Sun Y, Yu H, Zheng D, Cao Q, Wang Y, Harris D, et al. Sudan Black B reduces autofluorescence in murine renal tissue. Arch Pathol Lab Med 2011;135:1335-42.
DOI: https://doi.org/10.5858/arpa.2010-0549-OA
Kajimura J, Ito R, Manley NR, Hale LP. Optimization of single- and dual-color immunofluorescence protocols for formalin-fixed, paraffin-embedded archival tissues. J Histochem Cytochem 2015;64:112-24.
DOI: https://doi.org/10.1369/0022155415610792
Erben T, Ossig R, Naim HY, Schnekenburger J. What to do with high autofluorescence background in pancreatic tissues - an efficient Sudan black B quenching method for specific immunofluorescence labelling. Histopathology 2016;69:406-22.
DOI: https://doi.org/10.1111/his.12935
Wizenty J, Ashraf MI, Rohwer N, Stockmann M, Weiss S, Biebl M, et al. Autofluorescence: A potential pitfall in immunofluorescence-based inflammation grading. J Immunol Methods 2018;456:28-37.
DOI: https://doi.org/10.1016/j.jim.2018.02.007
Sutherland BW, Toews J, Kast J. Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions. J Mass Spectrom 2008;43:699-715.
DOI: https://doi.org/10.1002/jms.1415
Hoffman EA, Frey BL, Smith LM, Auble DT. Formaldehyde crosslinking: a tool for the study of chromatin complexes. J Biol Chem 2015;290:26404-11.
DOI: https://doi.org/10.1074/jbc.R115.651679
Yagi M, Toshima T, Amamoto R, Do Y, Hirai H, Setoyama D, et al. Mitochondrial translation deficiency impairs NAD+-mediated lysosomal acidification. EMBO J 2021;40:e105268.
DOI: https://doi.org/10.15252/embj.2020105268
Alex L, Tuleta I, Harikrishnan V, Frangogiannis NG. Validation of specific and reliable genetic tools to identify, label, and target cardiac pericytes in mice. J Am Heart Assoc 2022;11:e023171.
DOI: https://doi.org/10.1161/JAHA.121.023171
Weckbach LT, Grabmaier U, Uhl A, Gess S, Boehm F, Zehrer A, et al. Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis. J Exp Med 2019;216:350-68.
DOI: https://doi.org/10.1084/jem.20181102
Zhang Z, Neff L, Bradshaw AD, Fan H, Ye E, Richardson W, et al. Multimodal microscopy imaging of cardiac collagen network:Are we looking at the same structures? Proc.SPIE 2023;12355:16.
DOI: https://doi.org/10.1117/12.2650223
Piquereau J, Caffin F, Novotova M, Lemaire C, Veksler V, Garnier A, et al. Mitochondrial dynamics in the adult cardiomyocytes: Which roles for a highly specialized cell? Front Physiol 2013;4:102.
DOI: https://doi.org/10.3389/fphys.2013.00102
Kuznetsov A, Troppmair J, Sucher R, Hermann M, Saks V, Margreiter R. Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: Possible physiological role? Biochim Biophys Acta 2006;1757:686-91.
DOI: https://doi.org/10.1016/j.bbabio.2006.03.014
Weber KT. Cardiac interstitium in health and disease: The fibrillar collagen network. J Am Coll Cardiol 1989;13:1637-52.
DOI: https://doi.org/10.1016/0735-1097(89)90360-4
Karpishin T. Reducing tissue autofluorescence. Biotechniques 2018;64:131.
DOI: https://doi.org/10.2144/btn-2017-0117
Sakr N, Glazova O, Shevkova L, Onyanov N, Kaziakhmedova S, Shilova A, et al. Characterizing and quenching autofluorescence in fixed mouse adrenal cortex tissue. Int J Mol Sci 2023;24:3432.
DOI: https://doi.org/10.3390/ijms24043432
Kuo M-H, Allis CD. In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in a chromatin environment. Methods 1999;19:425-33.
DOI: https://doi.org/10.1006/meth.1999.0879
Vasilescu J, Guo X, Kast J. Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry. Proteomics 2004;4:3845-54.
DOI: https://doi.org/10.1002/pmic.200400856
Werner DA, Huang CC, Aminoff D. Micro method for determination of borohydride with NAD+. Anal Biochem 1973;54:554-60.
DOI: https://doi.org/10.1016/0003-2697(73)90387-4
Avigad G. Reduction of nicotinamide adenine dinucleotides by sodium cyanoborohydride. Biochim Biophys Acta - Enzymol 1979;571:171-4.
DOI: https://doi.org/10.1016/0005-2744(79)90239-0
Jensen T, Holten-Rossing H, Svendsen IMH, Jacobsen C, Vainer B. Quantitative analysis of myocardial tissue with digital autofluorescence microscopy. J Pathol Inform 2016;7:15.
DOI: https://doi.org/10.4103/2153-3539.179908
Miller CE, Thompson RP, Bigelow MR, Gittinger G, Trusk TC, Sedmera D. Confocal imaging of the embryonic heart: How deep? Microsc Microanal 2005;11:216-23.
DOI: https://doi.org/10.1017/S1431927605050464
King KR, Aguirre AD, Ye Y-X, Sun Y, Roh JD, Ng RP, et al. IRF3 and type I interferons fuel a fatal response to myocardial infarction. Nat Med 2017;23:1481-7.
DOI: https://doi.org/10.1038/nm.4428
Ding Y, Lee J, Ma J, Sung K, Yokota T, Singh N, et al. Light-sheet fluorescence imaging to localize cardiac lineage and protein distribution. Sci Rep 2017;7:42209.
DOI: https://doi.org/10.1038/srep42209
Schnell SA, Staines WA, Wessendorf MW. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 1999;47:719-30.
DOI: https://doi.org/10.1177/002215549904700601
Viegas MS, Martins TC, Seco F, do Carmo A. An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues. Eur J Histochem 2007;51:59-66.
Terman A, Brunk UT. Lipofuscin: mechanisms of formation and increase with age. APMIS 1998;106:265-76.
DOI: https://doi.org/10.1111/j.1699-0463.1998.tb01346.x
Ethics Approval
the animal protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Medical University of South CarolinaSupporting Agencies
South Carolina IDeA Networks of Biomedical Research Excellence , National Institutes of Health , MTF Biologics Extramural Research Grant, South Carolina Translation Research Improving Musculoskeletal Health , National Science Foundation EPSCoR ProgramHow to Cite
Zhang, Z., Fan, H., Richardson, W., Gao, B. Z., & Ye, T. (2023). Management of autofluorescence in formaldehyde-fixed myocardium: choosing the right treatment. European Journal of Histochemistry, 67(4). https://doi.org/10.4081/ejh.2023.3812
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